Genomic polymorphism in the T-even bacteriophages.

We have compared the genomes of 49 bacteriophages related to T4. PCR analysis of six chromosomal regions reveals two types of local sequence variation. In four loci, we found only two alternative configurations in all the genomes that could be analyzed. In contrast, two highly polymorphic loci exhibit variations in the number, the order and the identity of the sequences present. In phage T4, both highly polymorphic loci encode internal proteins (IPs) that are encapsidated in the phage particle and injected with the viral DNA. Among the various T4-related phages, 10 different ORFs have been identified in the IP loci; their amino acid sequences have the characteristics of internal proteins. At the beginning of each of these coding sequences is a highly conserved 11 amino acid leader motif. In addition, both 5' and 3' to most of these ORFs, there is a approximately 70 bp sequence that contains a T4 early promoter sequence with an overlapping inversely repeated sequence. The homologies within these flanking sequences may mediate the recombinational shuffling of the IP sequences within the locus. A role for the new IP-like sequences in determining the phage host range is proposed since such a role has been previously demonstrated for the IP1 gene of T4.     

Primary structure of internal peptide VII of T-even bacteriophages

As first shown by Hershey (1955,1957), osmotic rupture of the head of bacteriophage T2 releases, together with the DNA, an acid-insoluble protein fraction and an acid-soluble peptide fraction. Subsequent chromatographic analysis of the acid-soludle fraction of bacteriophage T4-infected Escherichia coli resolved two peptides, designated II and VII, corresponding to the peptide fraction of T2 described by Hershey (Eddleman &; Champe, 1966). Both peptides were found to be composed largely of acidic residues with only a limited number of other amino acids. The amino acid compositions predicted minimum molecular weights of 3900 (33 residues) and 2700 (23 residues) for peptides II and VII, respectively, consistent with their retardation on Sephadex G25 (Champe &; Eddleman, 1967). The formation of these peptides in T4-infected cells parallels the synthesis of late proteins. Pulse-labelling experiments showed, however, that the internal peptides are derived from acid-insoluble precursors, suggesting the probable involvement of proteolytic cleavage in their formation. The role of these peptides, if any, in phage replication is unclear. Eddleman &; Champe (1966) suggested that they may be inactive remnants of a core protein whose elimination is required for assembly. Alternatively, Laemmli &; Favre (1973) have proposed that the internal peptides may function to facilitate packaging of the DNA in the phage head.     

Lysis gene t of T-even bacteriophages: evidence that colicins and bacteriophage genes have common ancestors.

The lysis gene t of the T-even-like bacteriophage K3 has been cloned and sequenced. The gene codes for a protein with a predicted molecular weight of 25,200. Expression of the complete lysis protein was impossible, but peptides complementing T4 amber mutants in t are described. No known lysis protein of other phages is homologous to protein T. Also, the Escherichia coli phospholipase A is different from protein T. CelB, the lysis protein of the colicin E2 operon, shows a similarity to protein T. Sequences of colicins A, E1, and E2 are related to gene 38 sequences, the gene preceding t and coding for the phage adhesin. A common origin for colicin genes and phage genes is discussed, and a protein region in colicins that is responsible for receptor recognition is predicted.     

Sedimentation coefficient of T-even bacteriophage DNA

The sedimentation coefficient of T2 phage DNA has been studied by zone centrifugation in sector-shaped preparative centrifuge tubes over a concentration range of 0.02–2.0 μg./ml. DNA. These results have been compared to a similar study in the analytical centrifuge of T4 DNA over the range of 0.50–5.75 μg./ml. DNA. A value for the sedimentation coefficient of 60.7 ± 1.8 S. was obtained by the first method and a value of 61.3 ± 1.5 S. by the second.     

Receptor-recognizing proteins of T-even type bacteriophages. The receptor-recognizing area of proteins 37 of phages T4 TuIa and TuIb

Escherichia coli phages of the T4 family (T4, TuIa, TuIb) recognize their cellular receptors by means of a C-terminal region of protein 37; a dimer of this polypeptide (1026 residues in T4) is located at the distal part of the long tail fibers. Virions of the T2 family use protein 38 (which is attached to the free end of protein 37) for this purpose. The corresponding areas of genes 37 belonging to TuIa and TuIb were cloned and sequenced. Comparison of the deduced protein primary structures, including those of T4 and lambda Stf (Stf most likely representing a subunit of the side tail fibers of phage lambda) showed that an area of 70 to 100 residues is characterized by very variable sequences, while the sequences of the adjacent 43 to 44 C-terminal residues as well as those upstream from the variable region are highly homologous. The variable regions are flanked and interrupted seven or eight times by the motif His-x-His-y, with x and y most often being Ser or Thr; furthermore, the locations of these repeated tetrapeptides are conserved. Using hybrid phages obtained by recombination of one phage with cloned fragments of gene 37 of another, it could be shown that the area of this gene encoding receptor specificity includes the variable area. The situation is analogous to the known receptor-recognizing region of proteins 38 belonging to the T2-type family, except that the repeating sequence is of a different nature. In T4, receptor specificity is coded for by 382 base-pairs of the 3'-end of the gene, starting exactly at the variable area. It was found that T4 can use the outer membrane protein OmpC or lipopolysaccharide as receptors with the same efficiency, and it is proposed that the 70 residues of the variable part of the protein serve to bind to both ligands.     

Transient electric birefringence of T-even bacteriophages. IV. T2L0 and T6 with extended tail fibers

Transient electric birefringence measurements of the bacteriophages T2L0 and T6 were performed under such conditions that the tail fibers are extended. The data obtained are compared to previously reported data for T4B. For all T-even phages the degree of extension of the tail fibers is a function of pH, ionic strength, and temperature. For T4B, much higher ionic strengths are needed than for T2L0 and T6 to accomplish complete tail-fiber extension. The rotational diffusion coefficients of the phages with fully extended fibers are equal to 120 ± 3 sec^?1, 132 ± 5 sec^?1, 157 ± 4 sec^?1 for T2L0, T4B, and T6, respectively. The respective optical anistropies are ? (2.66 ± 0.05) × 10^?4, and ? (3.07 ± 0.15) × 10^?4. The differences in the rotational diffusion coefficient and optical anisotropy arise because the conformation of the fully extended tail fibers is different for the three phages. The tail fibers of T2L0 project further into the solution (away from the head) than do those of T4B and T6. The apparent permanent dipole moments of T2L0 and T6 decrease with increasing ionic strength. This decrease is caused by the screening of the surface charges on the phage body by the counter-ions in the solution. The biological relevance of this decrease is illustrated by the fact that the adsorption rate of T6 phages to E. coli B bacteria shows a similar dependence of ionic strength. Evidence is pressented that the tail fibers may move more or less independently of the phage body when an electric field is applied to the suspension.     

Structure of DNA within three isometric bacteriophages.

This paper describes a model for the structure of DNA contained in three morphologically similar bacteriophages--T7, P22 and phiCd-1--based on the transient electric dichroism of intact phage. The reduced dichroism of each of the phages at perfect orientation is within the range +0.12 to +0.19. Assuming that the phage orientation axis is that which passes from the apex through the tail, the measured dichroism suggests that DNA is wrapped in closely packed, co-axial solenoids with the axis of the solenoids tipped 43.5 degrees +/- 2.5 degrees from the orientation axis of the phage. All three phages show a large permanent dipole moment, with respective values of 5600, 200,000 and 500,000 Debye for T7, phiCd-1 and P22. The radius of the equivalent sphere for the three phages calculated from the rotational relaxation time for the rise of dichroism is in agreement with birefringence and electron microscope observations. The circular dichroism spectra of all three bacteriophages indicate that the local DNA helicity is similar in each case.     

Transient electric birefringence of T-even bacteriophages. II. T4B in the presence of tryptophan and T4D.

Measurements of electric birefringence, sedimentation velocity, and biological adsorption rate are used to study the properties of bacteriophage T4B in the presence of excess tryptophan. The adsorption rate determined in borate buffer pH 9 (at 25°C) increases from 0.003 × 10^?8 ml min^?1 (0.025 M) to 0.130 × 10^?8 ml min^?1 (0.150 M). The Kerr coefficient, rotational diffusion coefficient, and the sedimentation coefficient of the phage are also dependent on buffer concentration and reach plateau values above 0.12 M given by K_sp = ?(275 ± 18) × 10^?9 OD^?1 cm² statvolt^?2, D_25,w = 133 ± 4 sec^?1, and s_20,w = 818 ± 11 S. From a comparison of electric birefringence measurements of T4B and T4D it is concluded that T4D and T4B (in the presence of excess tryptophan) exhibit a similar hydrodynamic behavior. The change in physical parameters is solely due to a shift in fiber configuration. At high buffer concentrations the fibers make an angle of approximately 3π/4 with the sheath and the permanent dipole moment is about 200,000 D. This dipole moment is roughly ten times as large as that of a phage particle with nonextended fibers. This difference may be due to a change in hydrodynamic center upon fiber extension or to the presence of positive charges on the fiber tips, or both. At intermediate buffer concentrations the phage population behaves as if it were monodisperse. Probably not all six fibers are extended under such conditions.     

On the nature of HNO2 and UV damages in T-even bacteriophages

Summary The interpretation of experiments with HNO₂-inactivated phage T4 published byHarm (1960) and of similar experiments with UV inactivated phages T2 and T4 (Dulbecco 1952,Harm 1956, andEpstein 1958) is discussed. Two alternative theories are used as possible interpretations of the HNO₂ experiments: (1) The injection damage theory holding that a fractionJ of the damages caused in a phage by HNO₂ treatment prevent participation of the phage in the infectious process. (2) The finite damage theory holding that the damages produced by HNO₂ in the genetic material are much larger (have a greater target length) than UV damages, and otherways applying to HNO₂ damages the same theory earlier applied to UV-damages (Barricelli 1956 and 1960).Experimental methods to decide between the two theories are indicated. In the conclusion it is shown that the injection damage theory is not valid for UV damages in T2 and T4. In this case the theory is ruled out by the results ofDulbecco's (1952),Harm's (1956) andEpstein's (1958) MR experiments.With 2 Figures in the TextThis investigation was supported by research grant RG-6980 from the Division of General Medical Sciences and C-4437 from the National Cancer Institute of the National Institutes of Health, U.S.A. Public Health Service.     

Transient electric birefringence of T-even bacteriophages. I. T4B in the absence of tryptophan and fiberless T4 particles

Measurements of the electric birefringence of suspensions of T4B in the absence of tryptophan and of fiberless T4 particles show that both kinds of particles are hydrodynamically equivalent. Their rotational diffusion coefficients corrected to 25°C and water viscosity (D_25,w) are 280 ± 9 sec^?1 and 295 ± 10 sec^?1, respectively. These corrected rotational diffusion coefficients are almost independent of buffer concentration and temperature. The sedimentation coefficient (s_20,w) of T4 B is equal to 1023 ± 12 S, a value which is likewise independent of buffer concentration. By analysis of the field strength dependence of the steady-state birefringence and by reversing pulse experiments it could be shown that the orientation in an electric field is largely due to a permanent dipole moment. This dipole moment is somewhat dependent on buffer concentration and amounts to about 24,000 debye for T4B and 95,000 debye for fiberless T4. An approximate calculation shows that the difference in dipole moment may be ascribed to positive charges on the fiber tip (at least ten per fiber), to negative charges along the fiber or (and) positive charges on the fiberless particle at those places where the fibers are attached in normal particles.     

2011年第一届牛津国际噬菌体大会介绍

1会议简介噬菌体是一类以原核生物(包括细菌、真菌、放线菌或螺旋体等)为宿主的病毒,它最早于1915年被发现,之后,d’Herelle在1917年再次发现了这种能裂解细菌的生物类群,并将其命名为"bacteriophage",简称"phage"。近年来随着细菌耐药形势的日益严峻,越来越多有远见     

Structural aberrations in T-even bacteriophage. VI. Molecular weight of DNA from giant heads

Cummings et al. (1973) reported that whenl-canavanine was chased from a T-even. bacteriophage-infected culture with its analog,l-arginine, a new type of aberrant particle was formed. These particles, which were termed “lollipops”, had giant heads as long as 44 normal head lengths, and were filled with DNA. We have now separated these particles into different size classes ranging from about three to 13 normal head lengths and measured the molecular weight of their DNA. The DNA released from intact phage particles by neutral or alkaline detergent lysis was characterized using a recently described biophysical technique which determines DNA molecular weight from solution viscoelasticity. The maximum DNA size correlated roughly with phage head length, indicating that these giant heads were often filled with single, long DNA molecules rather than with several normal-sized molecules. Many of the heads, however, must have contained several molecules, since a large amount of DNA of less than maximum size was present. In alkali the native molecules separated into single strands of approximately the same length as that of the native molecules.     

The influence of glucosylation on the renaturation rate of T4 phage DNA

The kinetic complexity of T4 phage DNA with different degrees of glucosylation was determined by studies of DNA renaturation. It was found that this parameter decreased with decreasing degree of glucosylation, and that the kinetic complexity of non-glucosylated DNA was in agreement with the known genome size of T4 phage. This observation indicates that no significant correction for differences in GC content is necessary in the determination of genome sizes from renaturation data.