Phage Φ80 Research Articles

Generation of DNA fragments by enzymatic cleavage at sites sensitive to denaturation

Fragments of bihelical bacteriophage Φ80 DNA have been generated by enzymatic cleavage at denaturation-sensitive sites. The DNA is exposed to subcritical denaturation conditions which are known to result in the generation of small single-stranded loops (Inman, R. B. (1967) J. Mol. Biol. 28, 103–116), presumably at loci enriched for A · T base pairs. The loops are “fixed” by reaction with formaldehyde. In rapid succession the unreacted formaldehyde is removed and the DNA is digested with the single strand-specific endonuclease from Neurospora crassa. Polyacrylamide agarose gel electrophoresis is used to monitor the reaction or isolate the DNA fragments. This procedure provides one approach to studying the chromosomal distribution of regions that are strongly biased toward A · T or G · C base pairs and also afford a flexible complement to the site-specific endonucleases in isolating and studying specific regions of the chromosome.

A colicin-tolerant mutant of Escherichia coli with reduced levels of cyclic AMP and a strong bias towards lambda lysogeny.

A colicin-tolerant mutant of Escherichia coli which is temperature-dependent for tolerance and unable to grow at 40° without the addition of salt, plates λ+ wild-type phage with a reduced frequency and very turbid plaques at 30°, failing to form any plaques at 40° even though the cells become phage infected. Phage mutants λcI, λcII, λcY42 and λvir form clear plaques and plate with equal efficiency on the mutant and its parent strain at both temperatures. The results show that over 95% of the phage infected cells become lysogenized after λ+ phage infection. In contrast, the temperate phage P1 formed normal turbid plaques with equal frequency on both bacterial strains, but phage ϕ80 forms normal turbid plaques at 30° and clear (non-lysogenic) plaques at 40°. An investigation of the mutant strain showed that it not only lacked a large molecular weight component from its cell envelope but also had reduced capacity to synthesize cyclic-3′,5′-AMP. It is proposed that the mutation(s) in the colicin-tolerant mutant gives rise to a number of alterations, one of which changes the cell envelope and leads to the reduction of intracellular cyclic AMP and the other giving rise to an altered RNA polymerase specificity. These alterations lead to a changed ratio of the products of the λ-genes involved in the control of lysogeny and give rise to a bias towards the lysogenic response. The cIII gene product of λ is not as essential to lysogeny as cII or cI and therefore possibly acts as an internal control of other λ functions (e.g. cro).

Comparative study of thermal inactivation of phage φ80 and lambda

The heat sensitivity of lambda and φ80, including their derivatives or phenotypically mixed phages, was studied. These analyses, performed in the presence of EDTA, indicated that phage particles of wild-type φ80 are more resistant to heat inactivation than those of wild-type λ. The rate constant of thermal inactivation is directly proportional to the DNA content of φ80 phage particle. This is similar to results obtained from study of lambda deletion mutants. Activation energies required to inactivate phage particles are approximately 70 kcal/particle for φ80 and 50 kcal/ particle for λ. The inactivation rates for phenotypically mixed phages are dependent on the protein coat.

The influence of temperate bacteriophage φ105 on transformation and transfection in [formula omitted

The frequency of transformation in derivatives of Bacillus subtilis 168 is drastically reduced when the bacteria are made lysogenic for bacteriophage φ105. This reduction in the frequency of transformation is not paralleled by a similar reduction in the frequency of: (a) transfection with DNA from bacteriophages φ29 and SP01, or (b) transduction by PBS1 and SP10. The results indicate that there are significant differences between the processes of transformation and transfection.

Morphogenesis of bacteriophage phi 80: identification of the cistron 13 product.

An in vitro complementation reaction leading to the assembly of bacteriophage phi80 tails from component proteins is described. Tail assembly occurs when a lysate of any mutant in cistron 13 is mixed with a second lysate of a mutant in any of the other cistrons involved in tail formation. Lysates of mutants that are blocked in tail formation contain phage heads that can unite with free tails to form infective particles. The rate of the complementation reaction shows little dependence upon temperature, suggesting that the assembly depends largely upon the kinetic encounter of the interacting components. The tail component missing in cistron 13 mutant lysates was purified approximately 55-fold and shown to be, at least in part, a protein having a molecular weight of approximately 22,000. This protein was also released from highly purified infective phi80 particles after osmotic shock followed by heattreatment, suggesting that it most probably is an integral structural protein of the phage tail. Lysates of mutants of bacteriophage lambda that are defective in tail formation were shown to contain a tail component identical with or similar to the phi80 cistron 13 product.

Growth of bacteriophage phi 105 and its deoxyribonucleic acid in radiation-sensitive mutants of Bacillus subtilis.

Growth of phage phi105 and its deoxyribonucleic acid (DNA) was studied in radiation-sensitive mutants of Bacillus subtilis. The recA gene is required for optimal prophage induction with mitomycin C and for infectivity of prophage DNA. rec B gene is required for marker rescue from mature DNA. The importance of bacterial genes for phage DNA activity seems to depend on phage DNA structure.

Genetic exchanges within prophage φ80 correlated with exchanges between host markers on either side

Four mutations of phage φ80 were mapped in phage crosses in the order am32 — am2 — h- c40. In prophage crosses the same markers were mapped in the order c40 — am32 — am2 — h indicating cyclic permutation as specified by the Campbell model. Genetic exchanges between prophage markers were correlated with exchanges between bacterial markers on either side in quantitative agreement with expectations based on a linearly inserted prophage.

Distribution of nucleotides in Escherichia coli DNA from φ80 transducing bacteriophage

Escherichia coli DNA in the neighborhood of the φ80 attachment site, including the tryptophan operon, an ochre suppressor gene ( su c ) and F lac DNA transposed into the same region, was analyzed. DNA isolated from the transducing phages φ80 pt 1, φ80 pt 0, φ80 dsu c and φ80 d 1 lac was fragmented by shear and fractionated with respect to buoyant density of mercury complexes in cesium sulphate. Bacterial and episomal DNA components were identified by hybridization experiments. A segment containing the tryptophan operon consists of DNA ranging in G + C content from 43 to 57%, and the operator proximal end is richer in G + C. The composition of a segment between su c and att 80 varies in the same range, with an average G + C content of 51%. A segment containing lactose genes is more homogeneous, with an average G + C content of 54%. A portion of F factor, recovered from φ80 d 1 lac, ranges in G + C content from 37 to 52%.

Isolation of Transducing Particles of ϕ80 Bacteriophage That Carry Different Regions of the Escherichia coli Genome

It has been possible to mate two strains harboring F-prime (F') factors and to isolate from such matings rare recombinants that behave as though the two episomes had fused. Thus, two genes not previously linked may be brought into close proximity. An F' factor carrying the attachment site for varphi80 was fused with one carrying the met-ppc-arg region of the chromosome. Lysogenization of such a strain, followed by induction, led to the isolation of varphi80arg(+) and varphi80met(+) transducing phages. This technique may be utilized as a general method for joining diverse bacterial genes to the genome of phage varphi80.

Mapping of prophage and mature deoxyribonucleic acid from temperate Bacillus bacteriophage phi 105 by marker rescue.

By using temperature-sensitive (ts) and suppressor-sensitive (sus) mutants, 11 essential genes have been identified in phage phi105. The order of the genes has been established in two- and three-factor crosses. The genes can be arranged in a linear order; this order is identical in the vegetative phage and in the prophage. One gene essential for phage deoxyribonucleic acid (DNA) synthesis has been found. Marker rescue from prophage and mature DNA, taken up by competent bacteria, was studied by superinfection with phage carrying one sus and one ts mutation. In prophage DNA, all single markers studied are rescued at similar frequencies. The frequency of co-rescue of two markers is proportional to the recombinational distance between the markers. Thus, colinearity between the genetic map and the position on the DNA molecule of those mutations used to establish the map is demonstrated. The results indicate that the recombination frequencies observed in vegetative crosses are a relative measure of the physical distance between markers. All single markers are not rescued at equal frequencies from mature DNA. The frequency of co-rescue of two markers is related to the recombinational distance only over a distance about one-fourth or less of the genetic map. Markers separated by 10% recombination, or more, are co-rescued at 5 to 10% of the frequency of rescue of single markers. Shearing of mature DNA into half-sized molecules reduces the efficiency by which single markers are rescued by a factor of 5 to 10. The results of experiments on co-rescue of two markers from half-sized mature DNA indicate a preferred break-point near the middle of the genetic map; the results are compatible with a nonpermuted sequence in mature DNA. It is pointed out and discussed that mature DNA exhibits several anomalies in marker rescue experiments.

Low-Frequency Rescue of a Genetic Marker in Deoxyribonucleic Acid from Bacillus Bacteriophage φ105 by Superinfecting Bacteriophage

Markers in gene L, which maps at the right end of the vegetative and prophage maps, are rescued at a strongly reduced frequency from mature phi105 deoxyribonucleic acid (DNA) by superinfecting phage but at high frequency from vegetative and prophage DNA. It is suggested that the ends of mature DNA are degraded when DNA is taken up by competent cells.

Characterization of ribonucleic acid transcribed in vitro on phage phi 80 deoxyribonucleic acid.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTCharacterization of ribonucleic acid transcribed in vitro on phage φ80 deoxyribonucleic acidToshio Okamoto, Masahiro Sugiura, and Mituru TakanamiCite this: Biochemistry 1970, 9, 18, 3533–3541Publication Date (Print):September 1, 1970Publication History Published online1 May 2002Published inissue 1 September 1970https://doi.org/10.1021/bi00820a006RIGHTS & PERMISSIONSArticle Views9Altmetric-Citations14LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (916 KB) Get e-Alerts Get e-Alerts

Specialized transduction of the galactose genes of Escherichia coli by phage φ81

Some properties of temperate coliphage φ81 were studied in comparison with those of phages φ80 and λ. Plaque morphology, latent period, and immunity characteristics of this phage are distinct from those of φ80 and λ. In contrast to λ, φ81 as well as φ80 is unable to form plaques at 42 °, but phage particle itself is resistant to heat treatment at 60 ° for 30 min. Heteroimmune curing was observed between φ81 and λ. Genetic mapping has located the prophage site ( att81) between galactose and biotin markers on the Escherichia coli chromosome, the probable gene order being gal- att81- attλ- bio. Phage φ81 was found to transduce specifically the galactose and biotin genes at a frequency of 10 −6 to 10 −7 and 10 −7 to 10 −8, respectively, per plaque-forming unit (low frequency transduction, LFT). Some of the Gal + transductants obtained produced high frequency transducing (HFT) lysates upon induction, and were often unstable and segregated out Gal − clones at high frequency. Gal + transducing phages that are capable of forming plaques (φ81 pg) were obtained from one of these transductants. Buoyant density of gal + transducing phage studied almost coincided with that of plaque-forming phage φ81.

Characterization of inducible bacteriophages in Bacillus licheniformis.

Two morphologically distinct and physically separable defective phages have been found in Bacillus licheniformis NRS 243 after induction by mitomycin C. One of them (PBLB) is similar to the defective phage PBSX of B. subtilis, which has a density of 1.373 g/cm(3) in CsCl and a sedimentation coefficient of 160S. PBLB incorporates into its head mainly bacterial deoxyribonucleic acid (DNA) which has a sedimentation coefficient of 22S and a buoyant density in CsCl of 1.706 g/cm(3). The other phage (PBLA) has a morphology similar to the temperate phage phi105 of B. subtilis; the head diameter is about 66 nm, and it possesses a long and noncontractile tail. PBLA has a density of 1.484 g/cm(3) in CsCl and the phage-specific DNA, which is exclusively synthesized after induction by mitomycin C, has a density of 1.701 g/cm(3). PBLA DNA is double-stranded and has a sedimentation coefficient of 36S, corresponding to a molecular weight of 34 x 10(6) to 35 x 10(6) daltons. The phage DNA has one interruption per single strand, giving single-stranded segments with molecular weights of 13 x 10(6) and 4 x 10(6) daltons. Common sequences between the two phage DNA species and with their host DNA have been demonstrated by DNA-DNA hybridization studies. Both phage particles kill sensitive bacteria. However, all attempts thus far to find an indicator strain to support plaque formation have been unsuccessful.

Studies on the in vitro assembly of bacteriophage phi 80 and phi 80-lambda hybrids.

Suppressor-sensitive (sus) mutants of bacteriophage phi80 defective in late functions were classified, by means of in vitro assembly tests, into two complementation groups: head donors and tail donors. Each group of mutants was subdivided, by means of two-factor crosses, into six cistrons. Deletion mapping revealed clustering of tail and also of head cistrons. The two clusters were located in the left arm of vegetative phi80 (the tail specifying cluster being distal). In vitro cross complementation between phi80 and lambda sus mutants revealed that whereas lambda heads could quite efficiently bind phi80 tails to form viable phage, the union of phi80 heads and lambda tails was very much less efficient. Deletion mapping of the phi80 sus mutants, using both phi80 and i(phi80)h(lambda) deletion lysogens indicated congruent gross gene arrangement in the two related bacteriophages.

Characterization of Temperate Bacillus Bacteriophage φ105

Temperate Bacillus phage phi105 is serologically unrelated to previously described virulent Bacillus phages. Phage phi105 is incapable of generalized transduction. Prophage phi105 is inducible with mitomycin C. Phage phi105 contains double-stranded deoxyribonucleic acid (DNA) with a molecular weight of about 25 x 10(7) as determined by band sedimentation and electron microscopy. The per cent guanine plus cytosine of phi105 DNA is 43.5 as determined by buoyant density in CsCl and by thermal denaturation. Phage phi105 DNA may contain complementary single-stranded ends.

Directed transposition of the arabinose operon: A technique for the isolation of specialized transducing bacteriophages for any Escherichia coli gene

An F- thr-ara ‡ ‡ Abbreviations used: lac, ara, gal, utilization of lactose, arabinose, galactose; his, pyr, pur, arg, thr, leu, pro, trp requirement for histidine, pyrimidine, purine, arginine, threonine, leucine, proline, tryptophan; T1 r, T1,5 r, resistance to phage T1, phages T1 and T5; att80, chromosome attachment site for φ80; Sm s, Sm r, sensitive and resistant to streptomycin; φ80d ara, φ80d lac, defective φ80 transducing phage carrying the ara region, the lac region; F TS, F factor with mutation rendering its replication temperature sensitive; φ80 h, φ80 host range; φ80 v, φ80 virulent; λ hφ80, λ—φ80 hybrid having λ immunity and φ80 host range and attachment specificity. episome has been inserted into the Escherichia coli chromosome at the T1 locus near the attachment site for bacteriophage φ80. From a strain carrying such an insertion, a φ80d ara transducing phage was isolated. The techniques used should be applicable for the isolation of specialized transducing phages for many E. coli genes.

Evidence that Bacillus subtilis bacteriophage SP02 is temperate and heteroimmune to bacteriophage phi-105.

Some characteristics of Bacillus subtilis phage SPO2 which show that it is a temperate phage are presented. Wild-type SPO2 forms turbid plaques, similar to those of other temperate phages. SPO2 lysogenic strains which are resistant to SPO2 can be isolated; these strains remain stable lysogens despite the fact that they can no longer adsorb SPO2. SPO2 lysogenic strains can be grown for many generations in SPO2 antiserum and remain lysogenic. Phage SPO2 plates on phi105 lysogens and phage phi105 plates on SPO2 lysogens; this indicates that SPO2 and phi105 are heteroimmune. Phage phi105 plates on an SPO2-resistant strain; this indicates that SPO2 and phi105 adsorb to different receptor sites on the bacterial surface.

Linked transformation of bacterial and prophage markers in Bacillus subtilis 168 lysogenic for bacteriophage phi 105.

Level of competence reached by Bacillus subtilis 168 lysogenic for temperate phage phi 105 was reduced compared to that reached by nonlysogenic cells. This effect was probably related to an alteration of the bacterial surface. Deoxyribonucleic acid extracted from phi 105 lysogenic bacteria was used to transform other lysogenic bacteria. About 25% linkage was found between the bacterial phe-1 marker and prophage marker ts N15. The order of a few prophage markers relative to phe-1 was established in three-factor crosses. The usefulness of this system for a study of the linkage between an integrated prophage genome and that of its host was discussed.

Mapping of a temperate bacteriophage active on Bacillus subtilis.

Bacteriophage phi105 is a temperate bacteriophage for Bacillus subtilis 168. Temperature-sensitive and plaque mutants of phi105 were isolated. The results of two- and three-factor crosses with these mutants suggest the vegetative map of phi105 to be circular. The location of prophage phi105 between bacterial markers phe-1 and ilvA1 was shown by means of PBS1 transduction. Five markers in the prophage were linearly ordered with respect to the bacterial markers. Linkage between bacterial and prophage markers was demonstrated in transformation experiments with deoxyribonucleic acid extracted from lysogenic bacteria. The data demonstrate that prophage phi105 is linearly inserted into the bacterial chromosome.