T4 Strain Research Articles

Temperature-induced differences in growth rate and fatty acid composition in two strains of Escherichia coli 15T−

The saturated/unsaturated fatty acid ratio of Escherichia coli 15T- decreases almost threefold as growth temperature decreases from 43 to 27 degrees C, wheras the ratio of a fast-growing mutant derived from 15T- changes only half as much. Strain 15T- experiences a 2.4-fold change in doubling time across this temperature range, but doubling time in the mutant changes 3.3-fold.

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.

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.

Nucleotide alterations in bacteriophage T4 serine transfer RNA that affect the conversion of precursor RNA into transfer RNA

We have determined the nucleotide sequences of the serine transfer RNAs that are specified by wild-type and psu1+ amber-suppressor strains of bacteriophage T4. The two transfer RNAs have the same sequence except for their anticodons, where NGA in the wild-type species is mutated to CUA in the psu1+ species (N is a modified residue of U). These mutations are believed to confer suppressor activity on the psu1+ serine tRNA. Three secondary mutants derived from psu1+ by loss of suppressor activity have been characterized. The mutants do not synthesize serine tRNA, although they do synthesize the large precursor RNA from which serine tRNA is normally derived. Nucleotide sequence determinations of the mutant precursor RNAs show that each one differs from the psu1+ species by a nucleotide substitution that occupies either the TΨC loop (one mutant) or the adjoining base pair of the stem supporting the TΨC loop (two mutants) in the usual cloverleaf arrangement of the serine tRNA molecule. The mutant precursor RNAs are defective in the first of three steps normally associated with the conversion of precursor RNA into serine tRNA. The mutations may directly cause this defect, or the defect may result from the lack of modified nucleotides that also characterizes the mutant precursor RNAs.

Nucleotide alterations in the bacteriophage T4 glutamine transfer RNA that affect ochre suppressor activity

We have determined the nucleotide sequences of the glutamine transfer RNAs that are coded by wild-type and p su 2 + ochre-suppressor strains of bacteriophage T4. The two transfer RNAs have the same sequence except for their anticodons, where NUG in the wild-type species is mutated to NUA in the p su 2 + species (N is a modified residue of U). This mutation is believed to confer suppressor activity on the p su 2 + glutamine tRNA. Three mutants derived from p su 2 + by loss of suppressor activity have been characterized with respect to their sequence alterations. Each mutant specifies a transfer RNA differing from the p su 2 + species by a nucleotide substitution that occupies a base-paired region in the cloverleaf arrangement of the molecule. The mutants synthesize a reduced amount of tRNA that is defective in nucleotide modifications and processing at the 5′ and 3′ termini.

Conditionally lethal mutants of bacteriophage T4 defective in production of a transfer RNA

Mutants of bacteriophage T4 have been isolated that grow normally on EscheriChia coli B/5 but are unable to grow on a strain of E. coli (CT439) isolated from nature and previously shown to restrict growth of transfer RNA-deficient strains of T4. These mutants define five complementation groups, two of which affect the production of bacteriophage-coded tRNAs. One gene appears to affect the appearance of several tRNAs. The second gene (defined by the mutant HA1) is apparently the structural gene for a single tRNA species δ. Mutant HA1 produces no mature δ tRNA while revertants of HA1 that have regained the ability to grow on strain CT439 now make this tRNA in amounts correlated with the ability of the various revertants to grow on the restrictive host. The δ tRNAs made by several revertants of HA1 have been compared to T4 wild-type δ by two-dimensional fingerprint analyses of panreatic and T 1ribonuclease products. These comparisons have allowed tentative identification of the primary mutational alteration. On the basis of these results we conclude that the inability of mutant HA1 to grow on strain CT439 is due to a mutation in the structural gene of δ which interferes with its appearance.

Identity of genes coding for soluble and structural dihydrofolate reductases in bacteriophage T4.

By recombination between bacteriophage T4 wh2, a dihydrofolate reductaseless mutant, and T6, I have prepared T4 wh(T6), a T4 strain which codes for the T6-specific soluble dihydrofolate reductase. This strain has the heat sensitivity of T6, not T4, which provides direct evidence that the wh gene codes for both the soluble dihydrofolate reductase and the structural dihydrofolate reductase which is a constituent of T-even phage tail plates.

Frameshift mutation in the lysozyme gene of bacteriophage T4: Demonstration of the insertion of four bases and the preferential occurrence of base addition in acridine mutagenesis

Three mutant strains of bacteriophage T4, each of which carried two frameshift mutations were isolated and the amino acid sequences of their lysozyme proteins were analyzed. The changes of the primary structure were found to be from Ala 74 - Val- Arg 76 to Gly-Cys-Cys-Cys in eJ28 eJD2, from Ala 73 - Ala-Val-Arg- Gly 77 to Gly-Cys-Cys-(Cys, Gly) in eJD6 eJD2 and from Ala 73 to Val-Asp in eJD8 eJ28. From the codon assignments obtained from in vitro studies, these alterations in the amino acid sequences may be consistently explained by the following mutational events: (+ 4, −1) in eJ28 eJD2, (+1, −1) in eJD6 eJD2 and (−1, + 4) eJD8 eJ28. We have so far analyzed 15 pseudo-wild type strains of phage T4 which carried two or three frameshift mutations in the lysozyme locus and have demonstrated the addition and the deletion of one or two bases. In this communication we report that the acridine-type mutagen, proflavin, induces the addition of not only one and two bases but also of four bases. When we summarize the base changes due to frameshift mutations in T4 phage lysozyme and tryptophan synthetase of Escherichia coli, we find that acridines predominantly cause base additions and that spontaneous mutations induce additions and deletions of bases with almost equal frequency. The present analysis further indicates that the following codons are utilized by T4-infected E. coli in vivo: Ala, GCU; Val, GUU; Arg, CGU or CGC in the wild-type and Asp, GAU; Gly, GGC and GGA; Ala, GCU; Cys, UGU and UGC; Val, GUG in the mutant strains.

Interaction of morphogenetic genes of bacteriophage T4

Mutational events affecting growth of a bacteriophage T4 strain that was defective in a morphogenetic gene were investigated. The new mutations occurred in other of the morphogenetic genes, indicating interactions between different components of the phage assembly pathways. Bacteriophage T4D amber mutants defective in gene 18 (major structural gene for the tail sheath) or gene 37 (major tail fiber structural gene) were found that were unable to grow on Escherichia coli W3110 suC (containing the weak ochre suppressor, suG). Revertants could be selected that contained the original amber mutation in gene 18 or 37 and also a mutation at a second site, which permitted growth on W3110 suG. These second-site revertants could be distinguished from true revertants (to wild-type) by their inability to grow on an isogenic strain lacking a nonsense suppressor, W3110 su −. Similarly, for an amber mutant defective in gene 23 (major head membrane structural gene), second-site revertants could be found by selection on E. coli Ymel (ambersuppressing). When separated from their associated amber mutation, many second-site mutations were also amber. With an amber mutant in gene 18, secondary amber mutations were found in genes 5, 6, 7, 26, 27, 29 and 51 (tail baseplate genes); with an original amber mutation in gene 37, secondary amber mutations were found in genes 5, 6, 7, 8, 26, 27 and 29 (baseplate), gene 14 (head), and gene 15 (tail); and with an original amber mutation in gene 23, secondary ambers were found in gene 20 (head) and gene 25 (baseplate). The nonsense suppressor contained in W3110 suC or Ymel probably translates many amber codons with low efficiency. Thus a deficiency of product of a gene containing an amber mutation might result, especially if that gene product were used in several copies per phage particle. In such a case, the probability that any one phage particle would accumulate a complete complement of the deficient gene product should diminish rapidly. The hypothesis is developed that secondary amber mutations similarly lower the level of a second component involved in phage assembly. A balance of assembly processes that is essential for normal phage morphogenesis is thus restored.

Studies on photodynamic action. II. The action on Escherichia coli cells.

The photodynamic inactivation of Escherichia coli strain 15T-, B/r and B was studied using acridine orange and methylene blue as sensitizers. The sensitivity was strongly dependent on the growth phase, namely, the cells at the stationary phase were extremely resistant. Parallel to the sensitivity to ultraviolet or X-rays, strain B/r was less sensitive than strain B. There was no recovery of strain 15T- in a liquid medium (M9) after illumination in the presence of acridine orange. When chloramphenicol was added to this medium, the inactivation proceeded further during the incubation. The syntheses of DNA, RNA and protein were greatly inhibited.

A specific DNA methylase induced by bacteriophage 15

Analysis of the base composition of DNA extracted from various derivatives of Escherichia coli strain 15T − showed that during growth in the presence of bacteriostatic agents such as 5-aminouracil or 2-thiothymine, the content of 6-methylaminopurine increased while the content of 5-methylcytosine remained constant. These findings confirmed the earlier observations of Dunn & Smith (1958). This increase is apparently due to the induction of a new and different DNA methylase than that found in untreated bacteria. While the normal enzyme(s) produce both methylated products, the new enzyme methylates only adenine at a rate up to 20 times the normal. In vitro, enzyme from normal cells is without activity with homologous DNA; enzyme from cells grown in the presence of 5-aminouracil or 2-thiothymine utilizes E. coli 15T − DNA preferentially. The above changes are also brought about by treatment of cultures with ultraviolet light, mitomycin C or thymine starvation and re-addition, all procedures well known for their ability to induce lysogens of E. coli. Examination of lysates of treated cultures show that conditions leading to DNA methylation changes also cause induction of a seemingly defective phage in derivatives of strain 15. The new DNA methylase may be a phage-coded enzyme.

An in vitro transversion by a mutationally altered T4-induced DNA polymerase

In a reaction in which poly dC serves as template, highly purified T4 DNA polymerase has been shown to incorporate dTTP into poly dG, at a level ( T G ) of 10 −5 to 10 −6. The polymerase from a temperature-sensitive mutagenic strain of T4 with a mutation in the structural gene for DNA polymerase, substitutes T for G at a frequency about fourfold higher than the wild-type enzyme. The error frequency for both enzymes depends on dTTP and dGTP concentrations and can be increased about 5- to 20-fold by the substitution of manganese for magnesium in the reaction.

Studies of DNA replication in vivo. 3. Accumulation of a single-stranded isolation product of DNA replication by conditional mutant strains of T4.

The precedinig papers of this series1' 2 repotted that the iinitial product, of bacterial DNA replicationi is isolated as low-molecular-weight single-stranided DNA. This first initermediate is converted, in the cell, into the second initermediate whose structure wheni isolated appears to be that of a double-stranlded DNA moiety cointaining a small but significant single-strainded portioin as well as gaps in the newly synthesized stranid. Okazaki and his associates showed that an appreciable amount of newly syinthesized DNA is isolated as single-stranded DNA.' An important question at this time conieerninig the conversion between these two different stages of DNA replication is whether an unknown enzyme(s) is, directly or indirectly, involved. As a first step in seeking to answer this question, strains of bacteriophage harboring conditional lethal mutations in genes implicated in DNA replication were examined. This paper reports that bacteriophage T4 strains which conitain coinditional mutations of gene 41, under nonpermissive conditions, accumulate newly synthesized DNA that is isolated as sin-gle-strainded DNA.

The mechanism of lysis in phage T4-infected cells

Sensitive bacteria infected with mutant strains of phage T4 that lack active lysozyme do not lyse at the usual time, but oxygen uptake in these bacteria cease at the time lysis would normally occur. Normal lysis seems to be triggered by the cessation of metabolism and is accomplished by lysozyme. Superinfection that causes lysis inhibition prolongs the time during which oxygen uptake occurs.

A frame-shift mutation resulting in the deletion of two base pairs in the lysozyme gene of bacteriophage T4

The primary structure of the lysozyme of a strain of phage T4 carrying two frame-shift mutations ( eJD5 eJ201) has been found to differ from that of the wild-type strain by a sequence of four amino acids. One of the mutations has been shown to be due to the deletion of two bases and the other to the addition of two bases. Frame-shift mutations causing the addition and deletion of single bases and the addition of two bases have been identified previously. The codon GAA has been identified as coding for glutamic acid in the wild-type strain and AGG has been found to code for arginine in the double-mutant strain.

Exclusion of RNA bacteriophages and interference with their RNA replication by bacteriophage T4

After infection of Escherichia coli with an RNA phage, the ensuing replication of infectious RNA is temporarily interrupted when the culture is superinfected 20 minutes later with a strain of phage T4. Production of 28s viral RNA is likewise inhibited during this interruption. Most of the infectious RNA present at the time of T4 superinfection subsequently loses its infectivity. Since synthesis of double-stranded RNA is not inhibited, the lost RNA may be used as template for this synthesis. The average sedimentation coefficient of this double-stranded RNA is smaller than that of normal replicative intermediate. Addition of actinomycin D or chloramphenicol before T4 superinfection prevents the interruption. On the other band, maturation of RNA-phage particles continues for almost 10 minutes after T4 superinfection and this maturation requires continued protein synthesis. Simultaneous mixed infection with T4 and RNA phage inhibits incorporation of the parental RNA of the latter into double-stranded replicative form and completely excludes the production of RNA-phage progeny in more than 95% of the T4-infected bacteria. Both viable T4 and T4 mutants that give abortive infection interfere with RNA phages. The possible connections between the effects reported here and the interference by T4 with bacterial nucleic acid metabolism are discussed.

EFFECT OF THYMINE STARVATION ON CHROMOSOME TRANSFER DURING CONJUGATION IN <i>ESCHERICHIA COLI</i>

Two Hfr strains which had recieved the thymine requiring mutation of strain 15T-, but the colicinogenic factor were isolated. Mating experiments with these Hfr's revealed that thymine starvation of Hfr cells inhibits chromosome transfer. Uracil and histidine double starvation in Hfr cells, rather than being inhibitory, protects them from the spontaneous interruption of the process. The inhibitory effect of thymine starvation is differentially active, the more distant from the origin of an Hfr chromosome a given genetic marker is located, the more strongly transfer of that marker is inhibited. This inhibitory effect is (at least, partially) reversible.

Thymine incorporation and metabolism by various classes of thymine-less bacteria.

SUMMARY: Escherichia coli strain 15T- yielded 109 organisms from 1 μg. thymine and growth was continuous, not diphasic. Aminopterin-selected thymine-less bacteria, on the other hand, required 20-25 μg. thymine/ml. to sustain growth, although thymine incorporation was the same as for strain 15T-, approximately 10-9 μg./bacterium. In dearth of thymine the aminopterin-derived auxotrophs underwent thymine-less death (like strain 15T-), and in 5 μg. thymine/ml., after some initial growth, also underwent thymine-less death, although thymine uptake and DNA synthesis continued at a low rate. In 20 μg. thymine/ml. (and above) growth was diphasic. From these auxotrophs were derived, presumably the result of a second mutation, strains similar to strain 15T-. These differed from strain 15T-, however, as follows: they required 2 μg. thymine/ml. to initiate growth, they formed less thymidine phosphorylase, and they did not yield a particular class of revertant characteristic of strain 15T-. The aminopterin-derived auxotrophs were a thousand times more sensitive to inhibition by cytidine and uridine than were their double mutants or strain 15T-. This was the only trait discovered to correlate with their high thymine requirement. The high thymine requirement appeared not to be due to a permeability defect. Several classes of revertants were obtained from thymine-less bacteria. The majority regained thymidylate synthetase simultaneously with loss of ability to incorporate thymine efficiently. One class from strain 15T- was unique: it regained thymidylate synthetase without losing thymine in-corporation, and represented the one exception to the rule of mutual exclusion between these two traits. The aminopterin-derived auxotrophs were a distinctive, stable, and remarkably uniform class of thymine-less bacteria. They emphasized the uniqueness of strain 15T-, and illustrated the dual differentiation from a wild type possessed by their double mutants and also by strain 15T-.

The integrity of deoxyribonucleic acid extracted from Escherichia coli 15T- after thymine-less death

1. The integrity of DNA extracted from Escherichia coli strain 15T(-) after thymine-less death was examined by studying the effects of treatment with aqueous alkali on the solubility in dilute acids and by viscosity and ultracentrifugal measurements, some of which were designed to detect single-strand breaks or inter-strand cross-links. None of the results showed that there was any modification or damage associated with thymine-less death.

Phage Growth and Deoxyribonucleic Acid Synthesis inEscherichia coliInfected by a Thymine-Requiring Bacteriophage

Mathews, Christopher K. (Yale University, New Haven, Conn.). Phage growth and deoxyribonucleic acid synthesis in Escherichia coli infected by a thymine-requiring bacteriophage. J. Bacteriol. 90:648-652. 1965.-Cultures of Escherichia coli B infected with a mutant strain of phage T4 which cannot induce the formation of thymidylate synthetase produce deoxyribonucleic acid (DNA) at about two-thirds the rate of cultures infected with the parent strain. Under certain conditions the yield of viable phage observed with the mutant is one-third of that brought about by the wild-type strain. Addition of thymine increases both DNA synthesis and phage production in cells infected by the mutant. It is suggested that the ability to induce thymidylate synthetase formation in infected cells confers a selective advantage on the wild-type strain.