Properties of condensed bacteriophage T4 DNA isolated from Escherichia coli infected with bacteriophage T4.

Methods developed for isolating bacterial nucleoids were applied to bacteria infected with phage T4. The replicating pool of T4 DNA was isolated as a particle composed of condensed T4 DNA and certain RNA and protein components of the cell. The particles have a narrow sedimentation profile (weight-average s=2,500S) and have, on average, a T4 DNA content similar to that of the infected cell. Their dimensions observed via electron and fluorescence microscopy are similar to the dimensions of the intracellular DNA pool. The DNA packaging density is less than that of the isolated bacterial nucleoid but appears to be roughly similar to its state in vivo. Host-cell proteins and T4-specific proteins bound to the DNA were characterized by electrophoresis on polyacrylamide gels. The major host proteins are the RNA polymerase subunits and two envelope proteins (molecular weights, 36,000 and 31,000). Other major proteins of the host cell were absent or barely detectable. Single-strand breaks can be introduced into the DNA with gamma radiation or DNase without affecting its sedimentation rate. This and other studies of the effects of intercalated ethidium molecules have suggested that the average superhelical density of the condensed DNA is small. However, these studies also indicated that there may be a few domains in the DNA that become positively supercoiled in the presence of high concentrations of ethidium bromide. In contrast to the Escherichia coli nucleoid, the T4 DNA structure remains condensed after the RNA and protein components have been removed (although there may be slight relaxation in the state of condensation under these conditions).     

Demonstration of pyrimidine dimer-DNA glycosylase activity in vivo: bacteriophage T4-infected Escherichia coli as a model system.

An approach to the detection of pyrimidine dimer-DNA glycosylase activity in living cells is presented. Mutants of Escherichia coli defective in uvr functions required for incision of UV-irradiated DNA were infected with phage T4 denV+ or denV- (defective in the T4 pyrimidine dimer-DNA glycosylase activity). In the former case the denV gene product catalyzed the incision of UV-irradiated host DNA, facilitating the subsequent excision of thymine-containing pyrimidine dimers. Isolation of these dimers from the acid-soluble fraction of infected cells was achieved by a multistep thin-layer chromatographic system. Exposure of the dimers to irradiation that monomerizes pyrimidine dimers (direct photoreversal) resulted in the stoichiometric formation of free thymine. Thus, in vivo incision of UV-irradiated DNA dependent on a pyrimidine dimer-DNA glycosylase can be demonstrated.     

Excision of pyrimidine dimers in toluene-treated Escherichia coli.

Toluene-treated cells were used for examining excision of pyrimidine dimers in Escherichia coli strains W3110, DM845 (uvrA-), P3478 (polA-), and KS5064 (polAex1). Excision occurring in toluene-treated cells is rapid, adenosine 5'-triphosphate dependent, and requires the uvrA gene function. In strains lacking either the polymerizing or 5' leads to 3' exonucleolytic activity of deoxyribonucleic acid polymerase I, excision does occur. However, both in vivo and in vitro, the excision in such strains is initially slower than wild type.     

Bacteriophage-induced functions in Escherichia coli K (lambda) infected with rII mutants of bacteriophage T4

Rutberg, Blanka (Karolinska Institutet, Stockholm, Sweden), and Lars Rutberg. Bacteriophage-induced functions in Escherichia coli K(lambda) infected with rII mutants of bacteriophage T4. J. Bacteriol. 91:76-80. 1966.-When Escherichia coli K(lambda) was infected with rII mutants of phage T4, deoxycytidine triphosphatase, one of the phage-induced early enzymes, was produced at initially the same rate as in r(+)-infected cells. Deoxyribonuclease activity was one-third to one-half of that of r(+)-infected cells. This lower deoxyribonuclease activity was observed also in other hosts or when infection was made with rI or rIII mutants. Presence of chloramphenicol did not allow a continued synthesis of phage deoxyribonucleic acid in rII-infected K(lambda). No phage lysozyme was detected nor was any antiphage serum-blocking antigen found in rII-infected K(lambda). It is suggested that the rII gene is of significance for the expression of phage-induced late functions in the host K(lambda).     

Model for bacteriophage T4 development in Escherichia coli

Mathematical relations for the number of mature T4 bacteriophages, both inside and after lysis of an Escherichia coli cell, as a function of time after infection by a single phage were obtained, with the following five parameters: delay time until the first T4 is completed inside the bacterium (eclipse period, nu) and its standard deviation (sigma), the rate at which the number of ripe T4 increases inside the bacterium during the rise period (alpha), and the time when the bacterium bursts (mu) and its standard deviation (beta). Burst size [B = alpha(mu - nu)], the number of phages released from an infected bacterium, is thus a dependent parameter. A least-squares program was used to derive the values of the parameters for a variety of experimental results obtained with wild-type T4 in E. coli B/r under different growth conditions and manipulations (H. Hadas, M. Einav, I. Fishov, and A. Zaritsky, Microbiology 143:179-185, 1997). A "destruction parameter" (zeta) was added to take care of the adverse effect of chloroform on phage survival. The overall agreement between the model and the experiment is quite good. The dependence of the derived parameters on growth conditions can be used to predict phage development under other experimental manipulations.     

Bypass of pyrimidine dimers in DNA of bacteriophage T4 via induction of primer RNA

Bacteriophage T4 has a third pathway for repair of damaged DNA besides excision repair and recombination repair. This pathway is a mechanism for the toleration of lesions rather than the repair of lesions. The substrate for this process is gapped DNA copied from a damaged template. Evidence indicates that these gaps are filled, giving rise to daughter strands that are sensitive to heat and to treatments with RNAase. These daughter strands subsequently serve as templates for DNA that is resistant to RNAase. This third pathway is dependent upon gene 41 (RNA-priming protein), gene uvsZ (function unknown) and gene 30 (polynucleotide ligase) and is presumed to consist of 4 steps: (1) induction of primer RNA opposite the lesion in the template; (2) elongation of primers by DNA polymerase; (3) ligation of daughter-strand fragments, without removal of primer RNA; (4) replication of DNA carrying RNA sequences, giving homogeneous DNA strands. We have called this process 'Re-initiation repair'.     

Two-component nature of bacteriophage T4 receptor activity in Escherichia coli K-12.

Escherichia coli bacteriophage T4 uses the lipopolysaccharide of the outer cell envelope membrane as a receptor. Lipopolysaccharide from E. coli K-12 required a major outer membrane protein, polypeptide Ib, for phage inactivation.     

Postinfection control by bacteriophage T4 of Escherichia coli recBC nuclease activity.

Infection by bacteriophage T4 has previously been shown to cause a rapid inhibition of the host recBC DNase, an ATP-dependent DNase that is required for genetic recombination in Escherichia coli. We report here the partial purification of a protein ("T4 rec inhibitor") from extracts of T4-infected cells and some characteristics of the in vitro inhibition reaction with purified inhibitor and recBC nuclease. This inhibitory activity could not be purified from extracts of uninfected E. coli. Both the ATP-dependent exonuclease and DNA-dependent ATPase activities of recBC DNase are inhibited by T4 rec inhibitor. Experiments suggest that the inhibitor interacts with the nuclease in a stoichiometric manner. The biological significance of this inhibition is discussed with respect to control reactions in phage-infected cells.