Escherichia coli endoribonucleases involved in cleavage of bacteriophage T4 mRNAs

The dmd mutant of bacteriophage T4 has a defect in growth because of rapid degradation of late-gene mRNAs, presumably caused by mutant-specific cleavages of RNA. Some such cleavages can occur in an allele-specific manner, depending on the translatability of RNA or the presence of a termination codon. Other cleavages are independent of translation. In the present study, by introducing plasmids carrying various soc alleles, we could detect cleavages of soc RNA in uninfected cells identical to those found in dmd mutant-infected cells. We isolated five Escherichia coli mutant strains in which the dmd mutant was able to grow. One of these strains completely suppressed the dmd mutant-specific cleavages of soc RNA. The loci of the E. coli mutations and the effects of mutations in known RNase-encoding genes suggested that an RNA cleavage activity causing the dmd mutant-specific mRNA degradation is attributable to a novel RNase. In addition, we present evidence that 5'-truncated soc RNA, a stable form in T4-infected cells regardless of the presence of a dmd mutation, is generated by RNase E.     

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.     

Expression of the bacteriophage T4 denV structural gene in Escherichia coli.

The expression of the T4 denV gene, which previously had been cloned in plasmid constructs downstream of the bacteriophage lambda hybrid promoter-operator oLpR, was analyzed under a variety of growth parameters. Expression of the denV gene product, endonuclease V, was confirmed in DNA repair-deficient Escherichia coli (uvrA recA) by Western blot analyses and by enhancements of resistance to UV irradiation.     

Mutations affecting translation of the bacteriophage T4 rIIB gene cloned in Escherichia coli

Summary Mutant ribosome binding sites of the bacteriophage T4 rIIB gene, resident on an 873 bp DNA fragment, were cloned into a plasmid vector as in-frame fusions to a reporter gene, beta-galactosidase. The collection of mutations included changes in the region 5 to the Shine/Dalgarno sequence, a mutation of the Shine/Dalgarno sequence, the alternate initiation codons GUG, AUA and ACG, and mutants in which several closely spaced initiation codons compete with each other on the same mRNA. The results show that the secondary structure variations we have installed 5 to the Shine/Dalgarno sequence have little effect on translation. GUG is essentially as good an initiator of translation as AUG when they are assayed on separate messages, but is outcompeted at least 50-fold in the sequence AUGUG. AUA and ACG are poor start codons, and are temperature sensitive. The initiation codon pair AUGAUA, in which the AUG is only two nucleotides from the Shine/Dalgarno sequence, displays a novel cold-sensitive phenotype.     

Chemical synthesis of the T4 endonuclease V gene and its expression in Escherichia coli

A gene coding for T4 endonuclease V was constructed by joining chemically-synthesized oligodeoxyribonucleotides. This gene was inserted into an expression vector and expressed in E. coli.     

Escherichia coli detection by GFP-labeled lysozyme-inactivated T4 bacteriophage

Escherichia coli has been used as an indicator of the fecal contamination of water and food, identifying potential health hazards. In this study, an E. coli-specific bacteriophage, T4, was used to detect E. coli bacteria. The T4 phage small outer capsid (SOC) protein was used to present green fluorescent protein (GFP), an easily detectable marker protein, on the phage capsid. To inactivate phage lytic activity, we used the T4e(-) phage, which does not produce the lysozyme responsible for host cell lysis. Infection of E. coli K12 cells with the GFP-labeled T4e(-) phage (T4e(-)/GFP) enabled the visualization and distinction of E. coli K12 cells from T4 phage-insensitive cells, Pseudomonas aeruginosa. Prolonged incubation of E. coli K12 cells with the T4e(-)/GFP phage did not lead to cell lysis. Propagation of T4e(-)/GFP in host cells increased the intensity of green fluorescence, making the distinction of E. coli cells from other cells simple and effective. This method enables the rapid, conclusive quantitation of E. coli cells within an hour.     

Nonsense suppression context effects in Escherichia coli bacteriophage T4

Summary Nonsense suppression by supE44 has been examined in a collection of 14 T4 gene 22 and gene 23 UAG mutants, for which the precise gene location is known. In concordance with previous studies, UAG followed by a pyrimidine was inefficiently suppressed. However, among positions with similar 3 nucleotides, there was considerable variation in suppression efficiency. The competition between supE44 and Release Factor 1 (RF1) was also investigated following the introduction of a multicopy RF1 plasmid. An inverse relationship between the efficiency of suppression and RF1 competition was observed.     

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).     

Folate polyglutamates in T4D bacteriophage and T4D-infected Escherichia coli.

The folate compound which is a structural component of the Escherichia coli T-even bacteriophage baseplates, has been identified as the hexaglutamyl form of folic acid using a new chromatographic procedure (Baugh, C.M., Braverman, E. and Nair, M.G. (1974) Biochemistry 13, 4952-4957). It has also been found that the host cell contains a variety of polyglutamyl forms of folic acid. The major form is the triglutamate (about 50%) but small amounts of higher molecular weight folates including the octaglutamate (1.8%) have been identified. Upon infection with wild-type T4D bacteriophage there is a shift in the distribution of the folate compounds so that the folyl polyglutamyl compounds having the higher molecular weights are increased. Infection of E. coli with baseplate mutants of T4D containing an amber mutation in gene 28 resulted in the formation of significant amounts (over 7%) of folate compound(s) of molecular weight much higher than those observed either in uninfected cells or cells infected with wild-type T4D. It is suggested that the T4D gene 28 product functions to cleave glutamate residues from high molecular weight folyl polyglutamates to increase the availability of the folyl hexaglutamate for virus assembly.     

Physical mapping and cloning of bacteriophage T4 anti-restriction endonuclease gene.

We have proposed that the ability of T4 to produce non-glucosylated progeny after a single cycle of growth on a galU rglA rglB+ mutant of Escherichia coli is due to the initiation of the rglB+ function by a phage-coded, anti-restriction endonuclease protein. Based on this hypothesis, we screened T4 deletion mutants for failure to give a burst in this host. The absence of an arn gene in phage mutants lacking the 55.5- to 58.4-kilobase region is verified by their inability to protect secondary infecting non-glucosylated phage from rglB-controlled cleavage. A functional arn gene was cloned on plasmid pBR325, and the 0.8-kilobase insert DNA was shown to be homologous to the DNA missing in the arn deletion phage.     

Lysis of Escherichia coli cells induced by bacteriophage T4

Abstract Structural changes in the envelope of Escherichia coli cells accompanying their lysis from without by bacteriophage T4 have been studied. The hypothesis concerning the role of collapse of membrane potential and formation of periplasmic vesicles in the process of lysis from without has been advanced.     

Cloned genes for bacteriophage T4 late functions are expressed in Escherichia coli

We have constructed derivatives of plasmid pMB9 carrying EcoRI digestion fragments of bacteriophage T4 DNA that code for late gene functions. When Escherichia coli strains carrying these plasmids are infected with T4 amber mutants, burst sizes up to 30% of the wild-type level are obtained. Single burst experiments imply that the phage progeny result from complementation and do not depend on marker rescue. By electrophoretic and immunological techniques, we have established that the cloned T4 late genes are transcribed and translated in uninfected cells. A serum blocking assay has been used to quantitate the levels of one of the T4 gene products, gp11, before and after T4 infection. Uninfected cells containing the cloned T4 gene 11 DNA have 0.1% and mini cells have 1% of the gp11 levels per unit protein found in cells late after T4 wild-type infection. There is little or no additional gp10 and gp11 formed from the cloned genes after T4 infection.     

DNA injection during bacteriophage T4 infection of Escherichia coli.

The process of phage T4 DNA injection into the host cell was studied under a fluorescent microscope, using 4',6-diamidino-2-phenylindole as a DNA-specific fluorochrome. The phage DNA injection was observed when spheroplasts were infected with the artificially contracted phage particles having a protruding core. The DNA injection was mediated by the interaction of the core tip with the cytoplasmic membrane of the spheroplast. A membrane potential was not required for the process of DNA injection. On the other hand, DNA injection upon infection by intact noncontracted phage of the intact host cell was inhibited by an energy poison. Based on these observations, together with results from previous work, a model for the T4 infection process is presented, and the role of the membrane potential in the infection process is discussed.