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.     

Mutants of bacteriophage T4 deficient in the ability to induce nuclear disruption: shutoff of host DNA and protein synthesis gene dosage experiments, identification of a restrictive host, and possible biological significance.

The shutoff of host DNA synthesis is delayed until about 8 to 10 min after infection when Escherichia coli B/5 cells were infected with bacteriophage T4 mutants deficient in the ability to induce nuclear disruption (ndd mutants). The host DNA synthesized after infection with ndd mutants is stable in the absence of T4 endonucleases II and IV, but is unstable in the presence of these nucleases. Host protein synthesis, as indicated by the inducibility of beta-galactosidase and sodium dodecyl sulfate-polyacrylamide gel patterns of isoptopically labeled proteins synthesize after infection, is shut off normally in ndd-infected cells, even in the absence of host DNA degradation. The Cal Tech wild-type strain of E. coli CT447 was found to restrict growth of the ndd mutants. Since T4D+ also has a very low efficiency of plating on CT447, we have isolated a nitrosoguanidine-induced derivative of CT447 which yields a high T4D+ efficiency of plating while still restricting the ndd mutants. Using this derivative, CT447 T4 plq+ (for T4 plaque+), we have shown that hos DNA degradation and shutoff of host DNA synthesis occur after infection with either ndd98 X 5 (shutoff delayed) or T4D+ (shutoff normal) with approximately the same kinetics as in E. coli strain B/5. Nuclear disruption occurs after infection of CT447 with ndd+ phage, but not after infection with ndd- phage. The rate of DNA synthesis after infection of CT447 T4 plq+ with ndd98 X 5 is about 75% of the rate observed after infection with T4D+ while the burst size of ndd98 X 5 is only 3.5% of that of T4D+. The results of gene dosage experiments using the ndd restrictive host C5447 suggest that the ndd gene product is required in stoichiometric amounts. The observation by thin-section electron microscopy of two distinct pools of DNA, one apparently phage DNA and the other host DNA, in cells infected with nuclear disruption may be a compartmentalization mechanism which separates the pathways of host DNA degradation and phage DNA biosynthesis.     

Nuclear Disruption After Infection of Escherichia coli with a Bacteriophage T4 Mutant Unable to Induce Endonuclease II

Nuclear disruption after infection of Escherichia coli with a bacteriophage T4 mutant deficient in the ability to induce endonuclease II indicates that either (i) the endonuclease II-catalyzed reaction is not the first step in host deoxyribonucleic acid (DNA) breakdown or (ii) nuclear disruption is independent of nucleolytic cleavage of the host chromosome. M-band analysis demonstrates that the host DNA remains membrane-bound after infection with either an endonuclease II-deficient mutant or T4 phage ghosts.     

Development of Coliphage T5: Ultrastructural and Biochemical Studies

Electron microscopic studies of Escherichia coli infected with bacteriophage T5(+) have revealed that host nuclear material disappeared before 9 min after infection. This disappearance seemed to correspond to the breakdown of host deoxyribonucleic acid (DNA) into acid-soluble fragments. Little or no host DNA thymidine was reincorporated into phage DNA, except in the presence of 5-fluorodeoxyuridine (FUdR). Progeny virus particles were observed in the cytoplasm 20 min postinfection. Most of these particles were in the form of hexagonal-shaped heads or capsids, which were filled with electron-dense material (presumably T5 DNA). A small percentage (3 to 4%) of the phage heads appeared empty. On rare occasions, crystalline arrays of empty heads were observed. Nalidixic acid, hydroxyurea, and FUdR substantially inhibited replication of T5 DNA. However, these agents did not prevent virus-induced degradation of E. coli DNA. Most of the phage-specified structures seen in T5(+)-infected cells treated with FUdR or with nalidixic were in the form of empty capsids. Infected cells treated with hydroxyurea did not contain empty capsids. When E. coli F was infected with the DO mutant T5 amH18a (restrictive conditions), there was a small amount of DNA synthesis. Such cells contained only empty capsids, but their numbers were few in comparison to those in cells infected under permissive conditions or infected with T5(+). The cells also failed to lyse. These results confirm other reports which suggest that DNA replication is not required for the synthesis of late proteins. The data also indicate that DNA replication influences the quantity of viral structures being produced.     

Bacteriophage T4 Inhibits Colicin E2-Induced Degradation of Escherichia coli Deoxyribonucleic Acid I. Protein Synthesis-Dependent Inhibition

The deoxyribonucleic acid (DNA) of Escherichia coli B is converted by colicin E2 to products soluble in cold trichloroacetic acid; we show that this DNA degradation (hereafter termed solubilization) is subject to inhibition by infection with bacteriophage T4. At least two modes of inhibition may be differentiated on the basis of their sensitivity to chloramphenicol. The following observations on the inhibition of E2 by phage T4 in the absence of chloramphenicol are described: (i) Simultaneous addition to E. coli B of E2 and a phage mutated in genes 42, 46, and 47 results in a virtually complete block of the DNA solubilization normally induced by E2; the mutation in gene 42 prevents phage DNA synthesis, and the mutations in genes 46 and 47 block a late stage of phage-induced solubilization of host DNA. (ii) This triple mutant inhibits equally well when added at any time during the E2-induced solubilization. (iii) Simultaneous addition to E. coli B of E2 and a phage mutated only in gene 42 results in extensive DNA solubilization, but the amount of residual acid-insoluble DNA (20 to 25%) is more characteristic of phage infection than of E2 addition (5% or less). (iv) denA mutants of phage T4 are blocked in an early stage (endonuclease II) of degradation of host DNA; when E2 and a phage mutated in both genes 42 and denA are added to E. coli B, extensive solubilization of DNA occurs with a pattern identical to that observed upon simultaneous addition of E2 and the gene 42 mutant. (v) However, delaying E2 addition for 10 min after infection by this double mutant allows the phage to develop considerable inhibition of E2. (vi) Adsorption of E2 to E. coli B is not impaired by infection with phage mutated in genes 42, 46, and 47. In the presence of chloramphenicol, the inhibition of E2 by the triple-mutant (genes 42, 46, and 47) still occurs, but to a lesser extent.     

Control of Early Gene Expression of Bacteriophage T4: Involvement of the Host rho Factor and the mot Gene of the Bacteriophage

Many early mRNA species of bacteriophage T4 are not synthesized after infection of Escherichia coli in the presence of chloramphenicol. This has been interpreted as a need for T4 protein(s) to be synthesized to allow expression of some early genes, e.g., those for deoxycytidinetriphosphatase, deoxynucleosidemonophosphate kinase and UDP-glucose-DNA beta-glucosyltransferase. In the experiments described here, early mRNA of bacteriophage T4 was allowed to accumulate during chloramphenicol treatment. After the addition of rifampin to inhibit further RNA synthesis, and subsequent removal of chloramphenicol, the accumulated mRNA was permitted to express itself into measured enzyme activities. It was shown that the early mRNA species coding for deoxycytidinetriphosphatase and UDP-glucose-DNA beta-glucosyltransferase could be formed in the presence of chloramphenicol if the E. coli host cell carried a mutation in the structural gene for the RNA chain termination factor rho. This was interpreted to mean that T4 protein(s) with anti-rho activity is normally required for the expression of these two early genes. An altered rho-factor could not, however, relieve the need of phage protein synthesis for the formation of another early mRNA, that coding for deoxynucleosidemonophosphate kinase. In this case the mot gene of T4 seemed to be involved, since the primary infection of E. coli cells with the mot gene mutant tsG1 did not allow subsequent deoxynucleoside monophosphate kinase mRNA synthesis after wild-type phage infection in the presence of chloramphenicol. In control experiments, deoxynucleoside monophosphate kinase mRNA synthesis induced by wild-type phage superinfecting in the presence of chloramphenicol was facilitated by the primary infection with T4 phage containing an unmutated mot gene.     

Isolation and partial characterization of a bacteriophage T5 mutant unable to induce thymidylate synthetase and its use in studying the effect of uracil incorporation into DNA on early gene expression.

A mutant of phage T5 which is unable to induce thymidylate synthetase was isolated. T5 thy mutants synthesized less DNA than did wild-type T5, and the burst size of progeny phage was correspondingly reduced two- to threefold in thy+ Escherichia coli. No DNA or progeny phage were made in E. coli thy hosts grown in the absence of exogenous thymine. When the T5 thy mutation was recombined with a T5 dut mutation (unable to induce dUTPase), replication resulted in progeny which contained significant amounts of uracil in their DNA, and these phage failed to produce plaques unless the plating host was deficient in uracil-DNA glycosylase. T5 phage containing various amounts of uracil in their DNA were prepared and used to determine the effect of uracil on the induction of the early enzyme dTMP kinase. The presence of uracil in the parental DNA increased the rate of induction of this enzyme by about 2.5-fold. The T5 thy gene was mapped and is located near the T5 frd gene on the B region of the T5 genome.     

Isolation of a bacterial host selective for bacteriophage T4 containing cytosine in its DNA.

An Escherichia coli B strain, B834 galU56, has been isolated which supports growth of bacteriophage T4 with cytosine in its DNA while restricting growth of T4 with hydroxymethylcytosine. This host is partially deficient in uridine diphosphoglucose as determined by the ability of DNA isolated from T4 grown on it to accept glucose in an in vitro assay. In this mutant an intact rgl restriction system recognizes unglucosylated hydroxymethylcytosine residues in phage DNA, while the absence of a functional rB restriction function prevents degradation of unmodified DNA containing cytosine.     

Isolation and Partial Characterization of Bacteriophage T5 Mutants Deficient in the Ability to Induce Deoxynucleoside Monophosphate Kinase

Two mutants of bacteriophage T5 deficient in the ability to induce wild-type levels of deoxynucleoside monophosphate kinase were isolated and partially characterized. Both mutations were demonstrated to be in a structural gene for the kinase. One of the mutants, designated dnk 10, induces no detectable levels of dCMP, dGMP, or dTMP kinase activity. Because the mutant can successfully infect nonpermissive cells, phage-induced deoxynucleoside monophosphate kinase appears to be an unessential function for phage production. DNA synthesis in dnk 10-infected cells, however, is reduced to 30% of that observed in wild-type-infected cells; phage production is reduced by a comparable amount. The dnk mutation has been mapped and located on the "C" region of the T5 genetic map, 6.3 map units from the C1 locus.     

Partial Suppression of Bacteriophage T4 Ligase Mutations by T4 Endonuclease II Deficiency: Role of Host Ligase

Endonuclease II-deficient, ligase-deficient double mutants of phage T4 induce considerably more deoxyribonucleic acid (DNA) synthesis after infection of Escherichia coli B than does the ligase-deficient single mutant. Furthermore, the double mutant can replicate 10 to 15% as well as wild-type T4, whereas the single mutant fails to replicate. When the E. coli host is also deficient in ligase, the double mutant resembles the single mutant. The results indicate that host ligase can substitute for phage ligase when the host DNA is not attacked by the phage-induced endonuclease II.     

Inactivation of receptors for bacteriophage T5 during infection of Escherichia coli B.

During infection of Escherichia coli by bacteriophage T5, the cell surface receptors for the phage were inactivated so that they could not be isolated from the infected cells. A mutant of T5 that could only inject 8% of the T5 DNA did not cause the inactivation.     

利用重组噬菌体诱导表达的大肠杆菌表达系统

将编码噬菌体T7RNA聚合酶的基因克隆至噬菌体M13mpl8RFDNA中,置于lac启动子的控制之下,得到了可表达T7 RNA聚合酶的重组噬菌体M13HEP。利用该噬菌体感染含T7启动子表达质粒的宿主菌以提供T7RNA聚合酶,可以诱导T7启动子控制下的外源基因的表达。该噬茵体诱导表达系统已成功地表达了多种外源基因,特别是一些表达产物对宿主菌有毒性的基因。同时,通过细菌接合将F',因子从大脑杆菌XL1-blue转至大肠杆菌HMS174,构建了新的大脑杆菌菌株HMSl74F,,使得T7表达质粒构建、表达及单链制备可以在同一菌株中完成,得到了一个完整的T7表达系统。     

Fate of cloned bacteriophage T4 DNA after phage T4 infection of clone-bearing cells

Plasmid pBR322 replication is inhibited after bacteriophage T4 infection. If no T4 DNA had been cloned into this plasmid vector, the kinetics of inhibition are similar to those observed for the inhibition of Escherichia coli chromosomal DNA. However, if T4 DNA has been cloned into pBR322, plasmid DNA synthesis is initially inhibited but then resumes approximately at the time that phage DNA replication begins. The T4 insert-dependent synthesis of pBR322 DNA is not observed if the infecting phage are deleted for the T4 DNA cloned in the plasmid. Thus, this T4 homology-dependent synthesis of plasmid DNA probably reflects recombination between plasmids and infecting phage genomes. However, this recombination-dependent synthesis of pBR322 DNA does not require the T4 gene 46 product, which is essential for T4 generalized recombination. The effect of T4 infection on the degradation of plasmid DNA is also examined. Plasmid DNA degradation, like E. coli chromosomal DNA degradation, occurs in wild-type and denB mutant infections. However, neither plasmid or chromosomal degradation can be detected in denA mutant infections by the method of DNA--DNA hybridization on nitrocellulose filters.     

Phage and host genetic determinants of the specific anticodon loop cleavages in bacteriophage T4-infected Escherichia coli CTr5X

Anticodon loop cleavages of two host tRNA species occur in bacteriophage T4-infected Escherichia coli CTr5X, a host strain restricting phage mutants deficient in polynucleotide kinase (pnk) or RNA ligase (rli). The cleavage products accumulate with the mutants but are further processed in wt infection through polynucleotide kinase and RNA ligase reactions. Inactivating mutations in stp suppress pnk- or rli- mutations in E. coli CTr5X and, as shown here, also abolish the anticodon nuclease, implicating the stp product with this activity. We show also that there exist other suppressing mutations of a pnk- (pseT2) mutation that appear not to affect the anticodon nuclease and are not in stp. It has been shown that a single locus in E. coli CTr5X, termed prr, determines the restriction of pnk- or rli- mutants. A transductant carrying prr featured upon infection the anticodon nuclease reaction products, suggesting that prr determines the specific manifestation of this activity. However, prr does not encode the tRNA species that are vulnerable to the anticodon nuclease.     

Development of T7 phage and T7 phage containing apurinic sites in an exonuclease III, endonuclease IV double mutant of Escherichia coli.

The development of bacteriophage T7 was examined in an Escherichia coli double mutant defective for the two major apurinic, apyrimidinic endonucleases (exonuclease III and endonuclease IV, xth nfo). In cells infected with phages containing apurinic sites, the defect in repair enzymes led to a decrease of phage survival and a total absence of bacterial DNA degradation and of phage DNA synthesis. These results directly demonstrate the toxic action of apurinic sites on bacteriophage T7 at the intracellular level and its alleviation by DNA repair. In addition, untreated T7 phage unexpectedly displayed reduced plating efficiency and decreased DNA synthesis in the xth nfo double mutant.     

Purification and structures of recombining and replicating bacteriophage T7 DNA.

During the infection of Escherichia coli by bacteriophage T7, there is a gradual conversion of host DNA to T7 DNA. Recombination and replication occur during this time. We have devised a new way of examining the physical structures of the intermediates of these processes. It is based on the observation that there are no sites in T7 DNA susceptible to cleavage by the restriction endonuclease EcoRI. E. coli DNA, on the other hand, is susceptible to degradation by EcoRI. Thus, phage and host DNA can be separated by sucrose gradient centrifugation after treatment with EcoRI. Concatemeric T7 DNA contains a high proportion of branched, gapped, and whiskered structures. These appear to be intermediates of replication and recombination. This approach also monitors the conversion process from host to T7 DNA.     

Identification of the gene controlling the synthesis of the major bacteriophage T5 membrane protein.

After infection of Escherichia coli B by bacteriophage T5, a major new protein species, as indicated by polyacrylamide gel electrophoresis, appears in the cells' membranes. Phage mutants with amber mutations in the first-step-transfer portion of their DNA have been tested for their ability to induce membrane protein synthesis after they infect E. coli B. We have found that phage with mutations in the Al gene of T5 do not induce the synthesis of the T5-specific major membrane protein, whereas phage that are mutant in the A2 gene do induce its synthesis. We conclude that gene Al must function normally for T5-specific membrane protein biosynthesis to occur and that only the first 8% (first-step-transfer piece) of the DNA need be present in the cell for synthesis to occur.     

Effect of mutations of Escherichia coli K-12 chromosome on the integration of bacteriophage Mu introduced into cells by infection or conjugation

We present the detailed research on the previously described Escherichia coli K-12 Mud- mutants with impaired development of bacteriophage Mu. The ability of Mu phage DNA to penetrate into mutant cells on infection was shown. If introduced into the cells or combined with mud mutation by recombination, the prophage may be induced, which results in phage Mu lythic development and phage burst from mutant cells. In the course of conjugative transfer into the mutant cells, within a DNA fragment of the lysogenic donor chromosome, MupAp1 prophage is not inherited by recombinants. At the same time, Mu prophage deficient in genes A and B, whose products are required for transposition, is inherited by the mutant with the usual frequency. These data enable us to conclude that the mud mutations disturb the stage of conservative transposition which is connected with the insertion of the Mu prophage into the chromosome, after excision from the linear DNA introduced into the cells via infection or conjugation.     

Various characteristics of the anti-restriction mechanism in bacteriophage T5

Bacteriophage T5 is not confined by the restriction systems of the second type EcoRII and EcoRV. Bacteriophage T5 DNA is not modified by EcoRII and EcoRV methylases in vivo. The sites of recognition for restriction endonuclease EcoRV are mapped at 24.4; 57.6; 68.5; 70.2% of T5 DNA, while the sites at 5.1; 7.6% are recognized by EcoRII, the sites at 5.75; 6.0 and 6.5% are recognized by HpaI in FST. A high activity of restriction endonucleases EcoRI and EcoRV is demonstrated in crude extracts of E. coli B834 (RI) and E. coli B834 (RV) cells infected by bacteriophage T5. The simultaneous infection of E. coli B834 (RI) or E. coli B834 (RV) cells by the amber mutants of bacteriophage T5 and the suppressing phage lambda NM761 does not result in the protection of lambda DNA by the T5 anti-restriction mechanism. The presented data support the hypothesis that the anti-restriction mechanism of bacteriophage T5 is based on prevention of T5 DNA contacts with restriction enzymes by a specific phage protein.