Transmission of amber mutants of bacteriophage T4. III. Thermostability of the replication of amber mutants in cells of a non-permissive host is typical for the majority of phage tail genes

The article deals with determination of the spreading of the earlier discovered phenomenon of the temperature sensitivity of multiplication of T4 phage amber mutants. On the basis of the study of the dependence of multiplication of 50 amber mutants in 22 genes of T4 phage tail in the cells of non-permissive host on the incubation temperature in the range of 15-41 degrees C, the following conclusion is drawn: temperature sensitivity of multiplication of amber mutants appears to be gene-specific and is widely spread among T4 phage genes, i.e. in the case of amber mutants the burst size decreases, even for 14 tail genes, by several orders with the increase in incubation temperature. Temperature sensitivity of multiplication is typical of amber mutants in the genes whose proteins are either of small number in a phage particle (several molecules) or play the role of catalytic factors. Moreover, genes, amber mutants of which possess temperature sensitivity of multiplication, map in defined clusters.     

Transmission of amber mutants of bacteriophage T4. IV. The frequency of the phenomenon of temperature sensitivity of replication in non-permissive cells of amber mutants for the head genes

Dependence of multiplication of 42 single and double amber mutants in 16 phage head genes on the incubation temperature was studied in the cells of non-permissive host. For amber mutants in 6 head genes the birst size decreases by several orders, with the increase of the incubation temperature. Among amber mutants of the above mentioned genes, mutants in genes 4 and 65 can be distinguished as those with considerably large burst size at low temperature. Phage head genes form the groups, according to temperature sensitivity of multiplication of amber mutants. These groups, together with corresponding groups of phage tail genes, constitute common temperature-sensitive and non-sensitive gene groups on the phage genomic map.     

Transmission of amber mutants in phage T4. V. Positive effect of heat shock proteins on the replication of amber mutants in gene 31

The effect of growth of Escherichia coli BE, prior to infection, on multiplication of double amber mutant amN54-amNG71 in gene 31, mutant amN131-amNG114 in gene 26 and T4D wild-type at different temperatures has been studied. In the case of gene 31 mutant the increase in phage burst size, along with increase in growth temperature, was only observed. And this dependence seems to have the same character as the known dependence of growth temperature on cellular levels of heat shock proteins. Possibly, the product of gene 31 might be substituted to some extent by some heat shock protein. An antiserum against gene 31 protein immunoprecipitates heat shock protein, the molecular weight of which is close to the molecular weight of gene 31 protein. So, it seems likely that, in addition to supposed ability of this heat shock protein for functional substitution of gene 31 protein, these proteins might have some structural homology as well.     

The host-dependent restriction of growth of an RNA coliphage FI.

Phage FIC is a spontaneous host-dependent mutant of phage FI which is classified into the fourth group of RNA Escherichia coli phages (RNA coliphages). The mutant phage (FIC) grows normally in E. coli strain Q13 (permissive host), but poorly in strain A/lambda (non-permissive host) (9). Attempts to elucidate the regulatory mechanism of growth of the mutant phage in the non-permissive host revealed the following: (a) growth of the mutant phage was specifically restricted in E. coli strains that have certain suppressor genes for amber mutation; (b) the mutant phage RNA (FIC-RNA) could not produce progeny in the spheroplasts of the non-permissive host; (c) adsorption of the mutant phage to, and penetration of the mutant phage RNA into, the non-permissive host were normal; and (d) biosynthesis of the phage-specific late protein and RNA did not occur in the non-permissive host. Based on these results we conclude that phage FIC is a spontaneous azure-type mutant of the fourth group of RNA coliphage FI.     

The DNA-Delay Mutants of Bacteriophage T4

Mutants of phage T4 defective in genes 39, 52, 58-61, and 60 (the DNA delay or DD genes) are characterized by a delay in phage DNA synthesis during infection of a nonpermissive Escherichia coli host. Amber (am) mutants defective in these genes yield burst sizes varying from 30 to 110 at 37 C in E. coli lacking an am suppressor. It was found that when DD am mutants are grown on a non-permissive host at 25 C, rather than at 37 C, phage yield is reduced on the average 61-fold. At 25 C incorporation of labeled thymidine into phage DNA is also reduced to 3 to 10% of wild-type levels. Mutants defective in the DD genes were found to promote increased recombination as well as increased base substitution and addition-deletion mutation. These observations indicate that the products of the DD genes are necessary for normal DNA synthesis. The multiplication of the DD am mutants on an Su⁻ host at 37 C is about 50-fold inhibited if prior to infection the host cells were grown at 25 C. This suggests that a compensating host function allows multiplication of DD am mutants at 37 C in the Su⁻ host, and that this function is active in cells grown at 37 C prior to infection, but is inactive when the prior growth is at 25 C. Further results are described which suggest that the products of genes 52, 60, and 39 as well as a host product interact with each other.     

Conditionally lethal amber mutations in the dnaA region of the Escherichia coli chromosome that affect chromosome replication.

Three amber mutations, dna-801, dna-803, and dna-806, were isolated by localized mutagenesis of the dnaA-oriC region of the chromosome from an Escherichia coli strain carrying temperature-sensitive amber suppressors. When the mutations were not suppressed at 42 degrees C, the cells did not grow and DNA synthesis was arrested. They were very closely linked to each other and to the dnaA46 mutation. The mutant phenotype of each strain was converted to the wild type by infecting the mutants with specialized transducing phase lambda i21 dnaA-2 but not with lambda i21 tna. Derivatives of lambda i21 dnaA-2, each of which carried the amber mutation dna-801 dna-803, or dna-806, converted the dnaA mutant phenotype to Dna+ but did not convert rhe amber mutants to the wild-type phenotype. E. coli uvrB cells were irradiated with ultraviolet light and infected with each of these phage strains. An analysis of proteins synthesized in the cells revealed that two proteins with molecular weights of 50,000 and 43,000 were specified by lambda i21 dnaA-2 but not by lambda i21 tna. When the ultraviolet-irradiated cells did not carry an amber suppressor, the derivative phage with the amber mutation invariably failed to produce the 43,000-dalton protein, but when the host cell carried supF (tyrT), the protein was produced. The 50,000-dalton protein was unaffected.     

Growth and DNA synthesis of bacteriophage phi x174 in a dnaP mutant of Escherichia coli.

phi X174am3trD, a temperature-resistant mutant of bacteriophage phi X174am3, exhibited a reduced ability to grow in a dnaP mutant, Escherichia coli KM107, at the restrictive temperature (43 degrees C). Under conditions at which the dnaP gene function was inactivated, the amount and the rate of phi X174am3trD DNA synthesis were reduced. The efficiency of phage attachment to E. coli KM107 at 43 degrees C was the same as to the parental strain, E. coli KD4301, but phage eclipse and phage DNA penetration were inhibited in E. coli KM107 at 43 degrees C. It is suggested that the dnaP gene product, which is necessary for the initiation of host DNA replication, participates in the conversion of attached phages to eclipsed particles and in phage DNA penetration in vivo in normal infection.     

T-even-type bacteriophages use an adhesin for recognition of cellular receptors

Protein 38 of the Escherichia coli phage T4 is thought to be required catalytically for the assembly of the long tail fibers of this phage. It is shown that this protein of phage T2 and the T-even-type phage K3 and Ox2 act differently. It was found that NH2-terminal fragments of the protein, expressed from cloned fragments of gene 38 of phage K3, bind to gene 38 amber mutants of phage T2. Such phage or T2 gene 38 amber mutants, grown on a non-permissive host, possess a complete set of six tail fibers but are non-infectious. Both types of non-infectious phage could be repaired by incubation with an extract of cells harboring a cloned gene 38 of a host range mutant of phage K3, K3hx. The repaired phages had the host range of K3hx and not of T2. Immuno-electron microscopy showed that protein 38 is located at the free ends of the long tail fibers of phages T2, K3 and Ox2. The protein serves the recognition of the cellular receptor, i.e. it acts as an adhesin.     

The bacteriophage P4 alpha gene is the structural gene for bacteriophage P4-induced RNA polymerase

Two temperature-sensitive mutants of satellite phage P4 which do not synthesize P4 DNA at the nonpermissive temperature have been isolated. One of these phage is mutated in the P4 alpha gene. It complements a P4 delta mutant, but not a P4 alpha amber mutant; both mutants are phenotypically identical to alpha amber mutants in all properties studied. They synthesize P4 early proteins 1 and 2 as well as two additional P4-induced early proteins, 5 and 6, which are described here. P4 late proteins are not synthesized by these mutants and cannot be transactivated by helper phage P2. The mutants are unable to transactivate P2 late proteins from a P2 AB mutant. The P4 RNA polymerase activity which has been suggested to be involved in P4 DNA synthesis is not detected at the nonpermissive temperature. The P4 polymerase activity in partially purified extracts prepared from cells infected with the mutant at the permissive temperature is temperature sensitive. Reduced activity is found in vitro when these extracts are preincubated at 41 degrees C or assayed at temperatures higher than 37 degrees C. Thus, the P4 RNA polymerase is the product of the alpha gene. Temperature shift experiments show that the alpha gene product is required until late in the P4 cycle.     

Suppressors of gene 32 am mutants that specifically overproduce P32 (unwinding protein) in bacteriophage T4.

A gene 32 amber (am) mutant, amNG364, fails to grow on Escherichia coli Su3+ high temperatures, suggesting that the tyrosine residue inserted at the am codon by Su3+ leads to a temperature-sensitive gene 32 protein (P32). By plating amNG364 on E. coli Su3+ 45 degrees C, several pseudorevertants were found that proved to contain a suppressor (su) mutant in addition to the original am mutation. Crosses of two of these amNG364su strains to am+ phage indicated that the suppressors themselves are in or close to gene 32. Phage strains carrying either of the two su mutations, without amNG364, grew normally. When cells were infected by these su mutants and the proteins produced were examined by sodium dodecyl sulfate-gel electrophroesis, specific overproduction of P32 was found. Maximum overproduction compared to am+ phage was 6.6-fold for one su mutant and 2.4-fold for the other. Other proteins were produced in normal amounts and in normal time sequence. When amNG364su phage were allowed to infect E. coli S/6/5(Su-), the gene 32 am fragments produced were present at the same derepressed levels as in an infection by amNG364 without a suppressor. The suppressor mutations are interpreted as causing derepression of P32 by altering sites in this autogenously regulated protein involved in template recognition. Previously, specific derepression of gene 32 had only been shown using gene 32 conditional lethal mutants grown under restrictive conditions. We have shown that P32 can also be derepressed under permissive conditions, indicating that loss of P32 function is not necessary for specific derepression.     

A functional requirement for modification of the wobble nucleotide in the anticodon of a T4 suppressor tRNA

Temperature-sensitive mutants of E. coli have been isolated which restrict the growth of strains of bacteriophage T4 which are dependent upon the function of a T4-coded amber or ochre suppressor transfer RNA. One such mutant restricts the growth of certain ochre but not amber suppressor-requiring phage. Analysis of the T4 tRNAs synthesized in this host revealed that many nucleotide modifications are significantly reduced. The modifications most strongly affected are located in the anticodon regions of the tRNAs. The T4 ochre suppressor tRNAs normally contain a modified U residue in the wobble position of the anticodon; it has been possible to correlate the absence of this specific modification in the mutant host with the restriction of suppressor activity. Furthermore, the extent of this restriction varies dramatically with the site of the nonsense codon, indicating that the modification requirement is strongly influenced by the local context of the mRNA. An analysis of spontaneous revertants of the E. coli ts mutant indicates that temperature sensitivity, restriction of phage suppressor function, and undermodification of tRNA are the consequences of a single genetic lesion. The isolation of a class of partial revertants to temperature insensitivity which have simultaneously become sensitive to streptomycin suggests that the translational requirement for the anticodon modification can be partially overcome by a change in the structure of the ribosome.     

Specific Suppression of Mutations in Genes 46 and 47 by das, a New Class of Mutations in Bacteriophage T4D

Mutants in T4 genes 46 and 47 exhibit early cessation of deoxyribonucleic acid (DNA) synthesis ("DNA arrest") and decreased synthesis of late proteins and phage. In addition, mutants in genes 46 and 47 fail to degrade host DNA to acidsoluble products. It is shown here that this complex phenotype can be partially suppressed by mutation of a T4 gene external to genes 46 and 47 which has been named das for "DNA arrest suppressor." The das mutations were discovered as third-site mutations in spontaneous pseudorevertants of [46, 47] mutants; the pseudorevertants make small plaques on Escherichia coli B, whereas [46, 47] mutants make none. The [das, 46, 47] triple mutant exhibits increased DNA, late protein, and viable phage production compared to the double mutant [46, 47]. The [das, 46, 47] mutant also degrades more of the host DNA to acid-soluble products than does the [46, 47] mutant. The suppressor effect of the das mutation appears to be gene-specific: it suppresses both amber and temperature-sensitive mutations in genes 46 and 47 and does not suppress amber mutations in any of the other genes tested. The [das] single mutants make normal-sized plaques on E. coli B and exhibit nearly normal host DNA degradation, DNA synthesis, late protein synthesis, and viable phage production. The das mutations either define a new gene between genes 33 and 34 or are special mutations within gene 33.     

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.     

Mapping of functional sites on the primary structure of the contractile tail sheath protein of bacteriophage T4 by mutation analysis

In order to determine the functional roles of amino acid residues in gp18 (gp: gene product), the contractile tail sheath protein of bacteriophage T4, the mutation sites and amino acid replacements of available and newly created missense mutants with distinct phenotypes were determined. Amber mutants were also utilized for amino acid insertion by host amber suppressor cell strains. It was found that mutants that gave rise to a particular phenotype were mapped in a particular region along the polypeptide chain. Namely, all amino acid replacements in the cold-sensitive mutants (cs, which grows at 37 degrees C, but not at 25 degrees C) and the heat-sensitive mutant (hs, lose viability by incubation at 55 degrees C for 30 min) except for one hs mutant were mapped in a limited region in the C-terminal domain. On the other hand, all the temperature-sensitive mutants (ts, grow at 30 degrees C, but not at 42 degrees C) and carbowax mutants (CBW, can adsorb to the host bacterium in the presence of high concentrations of polyethylene glycol, where wild-type phage cannot) were mapped in the N-terminal protease-resistant domain, except for one ts mutant. The results suggested that the C-terminal region of gp18 is important for contraction and assembly, whereas the N-terminal protease-resistant domain constitutes the protruding part of the tail sheath.     

Identification and biosynthesis of the bacteriophage T4 mot regulatory protein.

The T4 mot gene regulates middle mode RNA synthesis in phage-infected cells. The mot gene product has been identified in two ways. (i) Infections with amber and temperature-sensitive mot mutants both lead to the disappearance of a number of protein bands on SDS-polyacrylamide gels. These are middle mode proteins whose synthesis depends on mot function. The mot protein disappears from such gels after infection with a mot amber mutant, but not with the mot missense mutant. (ii) This same protein is the only one to have a charge alteration when proteins from wild-type phage and mot missense mutant infections are compared by two-dimensional gel electrophoresis. Mot protein is basic and has a mol. wt. of 24 000. It migrates between the positions of gp 1 and gp IPIII on 15% SDS-polyacrylamide gels. Mot protein synthesis begins immediately after infection and continues until 4 min after infection at 30 degrees C, after which time it is strongly inhibited. This inhibition depends neither on T4 DNA synthesis nor on ADP ribosylation of the alpha subunits of the Escherichia coli RNA polymerase. The mot protein does not regulate its own biosynthesis. It is stable throughout the course of infection.     

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.     

Participation of the host protein(s) in the morphogenesis of bacteriophage P22

Summary Spontaneous mutants of S. typhimurium resistant to thiolutin are conditionally non-permissive for phage P22 development (Joshi and Chakravorty 1979). At 40° C non-infective phage particles are produced. Phage development in two nonpermissive hosts (18/MC4 and 153/MC4) has been studied in detail. The steps at which the phage morphogenesis is interfered with differ in the two mutants. The electron micrograph of the particles produced in the mutant 18/MC4 reveals the presence of normal-looking particles; these particles contain phage DNA, adsorb to the permissive host but fail to inject their DNA. The particles produced in the mutant 153/MC4 which fail to adsorb to the host are found to be tail fibre-less. These observations indicate the involvement of host protein(s) in phage P22 morphogenesis.     

Head morphologies in bacteriophage T4 head and internal protein mutant infections.

Escherichia coli infected with phage T4 mutants defective in synthesis of the three major internal proteins found in the phage head, IPI-, IPII-, IPIII-, or IP degrees (lacking all three) were examined in the electron microscope for head formation. Infection with IPI- or IPII- does not appear to induce increased aberrant head formation, whereas IPII- or IP degrees infections result in production of polyheads and viable phage. Multiple mutants of the early head formation genes 20, 21, 22, 23, 24, 31, 40 and IP degrees were constructed. Combination with IP degrees increases polyhead formation when head formation is not blocked at a more defective stage but results in a qualitative shift to lump formation in association with gene 22 mutants. Thin-sectioning studies show morphologically similar cores in amber 21 and 21am IP degrees tau particles. These morphological observations, genetic evidence for interaction between ts mutants in gene 22 and the IP mutants, and analysis of the protein composition of tau particles further support the idea that p22 and the internal proteins form an unstable assembly core necessary for an early stage of head formation (M. K. Showe and L. W. Black, 1973).     

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.     

Conditional lethal mutants of bacteriophage T4 unable to grow on a streptomycin resistant mutant of Escherichia coli.

Sixteen conditional lethal mutants of bacteriophage T4D have been isolated which grow on Escherichia coli CR63 (a su+ streptomycin-sensitive K12 strain) but are restricted by CR/s (a streptomycin-resistant derivative of CR63). These mutants have been given the prefix str. Four of these mutants are amber and 12 appear to be missense. Eleven of the 12 missense mutants appear to be "pseudo-amber" (i.e. they are restricted by a su- E. coli B strain but not by a su- K12 strain); the other missense mutant was not restricted by either B or K12. The str mutations mapped in 12 different genes. Most were clustered in a region of early genes (gene 56 to gene 47). Fifty-eight amber and 10 "pseudo-amber" mutants isolated previously for their inability to grow on E. coli B were tested for restriction by CR/s. All the amber mutants grew normally on CR/s, whereas all 10 "pseudo-amber" mutants were restricted by CR/s. This implies that the phenotype of the "pseudo-amber" mutants is the result of a ribosomal difference between the permissive host CR63 and the restrictive hosts B and CR/s. These str mutants should prove to be useful alternatives to amber mutants for genetic and biochemical studies of bacteriophage T4 and for studies of the E. coli ribosome. It should be possible ot isolate similar mutants in other bacteriophages provided that streptomycin resistant hosts are available.