Bacteriophage T4-related macromolecular synthesis under restriction of plasmid Rts1.

Rts1 is a plasmid which confers upon the host bacteria the capacity to restrict T4 bacteriophage growth at 32 degrees C but not at 42 degrees C. Pulse-labeling of phage-infected cells showed that Rts1 restricts the synthesis of T1 DNA. Despite efficient restriction of T4 phage growth and DNA synthesis, infected Escherichia coli 20SO harboring Rts1 synthesized both early and late T4 phage RNA. Synthesis of early T4 phage RNA under restrictive conditions (32 degrees C) was almost equal to that found under nonrestrictive conditions, and a lesser, but significant, amount of late T4 phage RNA was made in almost complete absence of T4 DNA synthesis. Moreover, very little, if any, T4 phage-coded lysozyme was detected in the infected E. coli 20SO/Rts1 at 32 degrees C, whereas normal amounts of lysozyme were present at 42 degrees C.     

SP62, a Viable Mutant of Bacteriophage T4D Defective in Regulation of Phage Enzyme Synthesis

SP62 is a mutant of bacteriophage T4D that was discovered because it produces fewer phage than the wild type in the presence of 5-fluorodeoxyuridine. In the absence of phage DNA synthesis, SP62 solubilizes host DNA slower than normal; this may explain the sensitivity to 5-fluorodeoxyuridine. In Escherichia coli B at 37 C in the absence of drugs, SP62 makes DNA at a normal rate and the kinetics of appearance of phage are nearly normal. Under the same conditions, SP62 produces T4 lysozyme (gene e) at a normal rate until 20 min, but then produces it at twice the normal rate until at least 60 min. It has long been known that, when T4 DNA synthesis is blocked (DNA⁻ state) in an otherwise normal infection, the synthesis of a number of early enzymes continues beyond the shutoff time of about 12 min seen in the DNA⁺ state, but still stops at about 20 min. We have termed the 12-min shutoff event S1 and the 20-min shutoff event S2. We show here that, in the DNA⁺ state, SP62 makes four early enzymes normally, i.e., S1 occurs. However, in the DNA⁻ state (where S1 is missing), SP62 continues to make dCTPase (gene 56), dCMP hydroxymethylase (gene 42), and deoxynucleotide kinase (gene 1) for at least an hour; this results in production of up to 13 times the normal level of dCTPase at 60 min after infection, or 6 times the DNA⁻ level. We conclude that SP62 is defective in the second shutoff mechanism, S2, for these three enzymes. In contrast, SP62 causes premature cessation of dTMP synthetase production in the DNA⁻ state; the result is a twofold underproduction of dTMP synthetase. Autoradiograms of pulse-labeled proteins separated by slab-gel electrophoresis in the presence of sodium dodecyl sulfate show that a number of other T4 early proteins, including the products of genes 45, 46, and rIIA, are synthesized longer than normal by SP62 in the DNA⁻ state. Few late proteins are made in the DNA⁻ state, but in autoradiograms examining the DNA⁺ state there is little or no effect of the SP62 mutation on the synthesis of T4 late or early proteins. Circumstantial evidence is presented favoring a role for the gene of SP62 in translation of certain mRNAs. At very high temperatures (above 43 C) in the absence of drugs, phage production, but not DNA synthesis, is much reduced in SP62 infections relative to wild-type T4 infections; this temperature sensitivity is greater on E. coli CR63 than on E. coli B. This property has facilitated recognition of the SP62 genotype and aided in complementation testing and genetic mapping. A later publication will provide evidence that SP62 defines a new T4 gene named regA, which maps between genes 43 and 62.     

A cytotoxic early gene of Bacillus subtilis bacteriophage SPO1.

Some of the early genes of Bacillus subtilis bacteriophage SPO1 were hypothesized to function in the shutoff of host biosyntheses. Two of these genes, e3 and e22, were cloned and sequenced. E22 showed no similarity to any known protein, while E3, a highly acidic protein, showed significant similarity only to other similarly acidic proteins. Each gene was immediately downstream of a very active early promoter. Each was expressed actively during the first few minutes of infection and was then rapidly shut off and its RNA rapidly degraded. An e3 nonsense mutation severely retarded the degradation of e3 RNA. Expression of a plasmid-borne e3 gene, in either B. subtilis or Escherichia coli, resulted in the inhibition of host DNA, RNA, and protein syntheses and prevented colony formation. However, the e3 nonsense mutation caused no measurable decrease in either burst size or host shutoff during infection and, in fact, caused an increased burst size at high multiplicities of infection. We suggest that e3 is one of several genes involved in host shutoff, that its function is dispensable both for host shutoff and for phage multiplication, and that its shutoff function is not entirely specific to host activities.     

Identification and preliminary characterization of a mutant defective in the bacteriophage T4-induced unfolding of the Escherichia coli nucleoid.

The nucleoids of Escherichia coli S/6/5 cells are rapidly unfolded at about 3 min after infection with wild-type T4 bacteriophage or with nuclear disruption deficient, host DNA degradation-deficient multiple mutants of phage T4. Unfolding does not occur after infection with T4 phage ghosts. Experiments using chloramphenicol to inhibit protein synthesis indicate that the T4-induced unfolding of the E. coli chromosomes is dependent on the presence of one or more protein synthesized between 2 and 3 min after infection. A mutant of phage T4 has been isolated which fails to induce this early unfolding of the host nucleoids. This mutant has been termed "unfoldase deficient" (unf-) despite the fact that the function of the gene product defective in this strain is not yet known. Mapping experiments indicate that the unf- mutation is located near gene 63 between genes 31 and 63. The folded genomes of E. coli S/6/5 cells remain essentially intact (2,000-3,000S) at 5 min after infection with unfoldase-, nuclear disruption-, and host DNA degradation-deficient T4 phage. Nuclear disruption occurs normally after infection with unfoldase- and host DNA degradation-deficient but nuclear disruption-proficient (ndd+), T4 phage. The host chromosomes remain partially folded (1,200-1,800S) at 5 min after infection with the unfoldase single mutant unf39 x 5 or an unfoldase- and host DNA degradation-deficient, but nuclear disruption-proficient, T4 strain. The presence of the unfoldase mutation causes a slight delay in host DNA degradation in the presence of nuclear disruption but has no effect on the rate of host DNA degradation in the absence of nuclear disruption. Its presence in nuclear disruption- and host DNA degradation-deficient multiple mutants does not alter the shutoff to host DNA or protein synthesis.     

“Host Shutoff” Function of Bacteriophage T7: Involvement of T7 Gene 2 and Gene 0.7 in the Inactivation of Escherichia coli RNA Polymerase

The "host shutoff" function of bacteriophage T7 involves an inactivation of the host Escherichia coli RNA polymerase by an inhibitor protein bound to the enzyme. When this inhibitor protein, termed I protein, was removed from the inactive RNA polymerase complex prepared from T7-infected cells by glycerol gradient centrifugation in the presence of 1 M KCl, the enzyme recovered its activity equivalent to about 70 to 80% of the activity of the enzyme from uninfected cells. Analysis of the activity of E. coli RNA polymerase from E. coli cells infected with various T7 mutant phages indicated that the T7 gene 2 codes for the inhibitor I protein. The activity of E. coli RNA polymerase from gene 2 mutant phage-infected cells, which was about 70% of that from uninfected cells, did not increase after glycerol gradient centrifugation in the presence of 1 M KCl, indicating that the salt-removable inhibitor was not present with the enzyme. It was found that the reduction in E. coli RNA polymerase activity in cells infected with T7(+) or gene 2 mutant phage, i.e., about 70% of the activity of the enzyme compared to that from uninfected cells after glycerol gradient centrifugation in the presence of 1 M KCl, results from the function of T7 gene 0.7. E. coli RNA polymerase from gene 0.7 mutant phage-infected cells was inactive but recovered a full activity equivalent to that from uninfected cells after removal of the inhibitor I protein with 1 M KCl. E. coli RNA polymerase from the cells infected with newly constructed mutant phages having mutations in both gene 2 and gene 0.7 retained the full activity equivalent to that from uninfected cells with or without treatment of the enzyme with 1 M KCl. From these results, we conclude that both gene 2 and gene 0.7 of T7 are involved in accomplishing complete shutoff of the host E. coli RNA polymerase activity in T7 infection.     

SP-10 bacteriophage-specific nucleic acid and enzyme synthesis in Bacillus subtilis W23.

Bacillus subtilis W23 was infected with a clear-plaque variant of SP-10 phage, namely, SP-10c. Exogenous thymidine was not incorporated into phage DNA (even in the presence of deoxyadenosine), nor was there any transfer of thymidine nucleotides from bacterial to viral DNA. The lytic program was unaffected by concentrations of 5-fluorodeoxyuridine sufficient to reduce bacterial DNA synthesis by greater than 95%. Although these data are consistent with the interpretation that thymidine nucleotides are excluded from phage DNA, formic acid digests of SP-10c DNA contained what appeared to be the four conventional bases; however, adenine and thymine were not recovered in equimolar yields. DNA-RNA hybridization and hybridization competition experiments were done. Synthesis of host RNA started to wane moments postinfection and stopped completely by 36 min. SP-10c coded for discrete classes of early and late RNA. The possibility of discrete subclasses of early RNA exists. Replication of the bacterial genome appeared to terminate 12 min postinfection. Degradation of the host DNA to acid-soluble material started at 36 min and, by the end of the latent period, greater than 90% of the host chromosome was hydrolyzed. Four apparent phage-coded enzymes have been identified. A di- and triphosphatase degraded dUTP, dUDP, dTTP, and dTDP (and, to a lesser extent, dCDP and d CTP) to the corresponding monophosphates; the enzyme had no apparent activity on dATP and dGTP. SP10c also coded for a DNA-dependent DNA polymerase, lysozyme, and a nuclease that degrades native bacterial DNA. Judging from the dependence of enzyme synthesis on the time of addition of rifampin (an inhibitor of the initiation of RNA synthesis), messengers for the di- and triphosphatase, as well as the nuclease, are transcribed from promoters that start to function 6 min postinfection. Promoters for polymerase and lysozyme did not become functional until 8 and 16 min postinfection, respectively.